THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 245, No. 20, Issue of October 25, PP. 5214-5222, 1070 Printed in U.S.A. The Interactions of Acetoacetate Decarboxylase with Carbonyl Compounds, Hydrogen Cyanide, and an Organic Mercurial* (Received for publication, June 2, 1970) ANNE P. AUTOR~ AND I. FRIDOVICH§ From the Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27706 SUMMARY 1. Hydrogen cyanide inhibited acetoacetate decarboxylase only when incubated with the enzyme in the presence of car-bony1 compounds. This resulted in an inhibitory syner- gism between hydrogen cyanide and carbonyl compounds. This synergism was used to investigate the comparative abilities of various carbonyl compounds to form Schiff’s bases at the active site of the enzyme. The order of effec- tiveness among the compounds tested was acetaldehyde > acetone > cyclohexanone > methyl ethyl ketone > 3-hexa- none > diethyl ketone. 2. The ability of hydrogen cyanide to inhibit the enzyme was optimal at pH 6 and decreased abruptly on both sides of this optimum. Although primarily an uncompetitive inhibi- tor of the enzyme, hydrogen cyanide caused perceptible de- creases in the slopes of the lines obtained by plotting kinetic data on reciprocal coordinates. Thus, raising the concen- tration of substrate increased the degree of inhibition caused by a given level of hydrogen cyanide more than could be ex- plained on the basis of combination of the inhibitor only with enzyme-substrate or enzyme-product complexes. 3. Continuous infusion of borohydride into well stirred and well buffered solutions of the enzyme resulted in no loss of activity unless carbonyl compounds, capable of forming Schiff’s base compounds at the active site, were present. The rate of inactivation of the enzyme, under these condi- tions, could be used as an index of the degree of saturation of the active sites by the carbonyl compound being tested and of the reactivity with borohydride of the Schiff’s base compounds so generated at the active site. Quantitative data were obtained in this way for acetone sulfonate, ace- tone, methyl ethyl ketone, and cyclohexanone. Nitrate was observed to protect the enzyme against inactivation by boro- hydride in the presence of acetone. 4. A number of diketones were compared for their abilities to inhibit the enzyme and the structural features essential for this action were discussed. P-Diketones, which inhibited the enzyme, generally did so with perceptible slowness which was accentuated by electron-withdrawing substituents ad- * The work reported herein was supported in full by Grant GM-10287 from the National Institutes of Health, Bethesda, Maryland. $ Predoctoral trainee of the National Institutes of Health, Bethesda, Maryland. Present address, Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48104. $ To whom all requests for reprints should be addressed. jacent to one of the carbonyl functions. The ability of ace- tylacetone to protect the enzyme against inactivation by borohydride, like its ability to inhibit the enzyme, developed slowly. 5. The reaction of acetoacetate decarboxylase with p-chlo- romercuriphenylsulfonate (CMS) was investigated both in terms of mercaptide formation and in terms of activity. One sulfhydryl group per subunit could be derivatized without loss of activity whereas reaction of a second sulfhydryl was associated with some inactivation. A third equivalent of CMS per subunit reached with an unidentified group to, cause additional inactivation. Amounts of CMS in excess of three per subunit had no further effects and the enzyme retained activity which was impervious to additional CMS. The native enzyme retained 70% of its activity after treat- ment with CMS whereas the fully activated enzyme retained only 50% of its activity after exposure to CMS. Inhibition by CMS was reversed by cysteine. The mechanism of acetoacetate decarboxylase has been shown to involve the intermediate formation of a Schiff’s base, formed by reaction of the carbonyl group of the substrate with the E-NH2 group of a specific lysine residue (I, 2). Since hydrogen cyanide is known to add across the double bond of azomethines (3-5) it was not surprising to observe that hydrogen cyanide inhibited this enzyme reversibly and with perceptible slowness and that this inhibition by cyanide developed only in the presence of substrate (1, 6). The ability of hydrogen cyanide to protect the enzyme against the substrate-dependent inactivation of the enzyme by borohydride (6) also supported this interpretation of its action. Indeed, several enzymes whose action involves Schiff’s base intermediates have similarly been found to be inhibited by hydrogen cyanide (7, 8). If hydrogen cyanide does inhibit acetoacetate decarboxylase by forming an adduct with a Schiff’s base intermediate at the active site, then it should aug- ment the inhibition caused by any carbonyl compound capable of forming such an azomethine. This synergism should be useful in detecting the ability of carbonyl compounds to react with the specific E-NH2 group at the active site of acetoacetate decarboxylase. Our conceptions of its action also predict that hydrogen cyanide should be an uncompetitive inhibitor of acetoacetate decarboxylase. Experiments which explore these 5214 by guest on January 25, 2020 http://www.jbc.org/ Downloaded from
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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 245, No. 20, Issue of October 25, PP. 5214-5222, 1070
Printed in U.S.A.
The Interactions of Acetoacetate Decarboxylase with Carbonyl Compounds, Hydrogen Cyanide, and an Organic Mercurial*
(Received for publication, June 2, 1970)
ANNE P. AUTOR~ AND I. FRIDOVICH§
From the Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27706
SUMMARY
1. Hydrogen cyanide inhibited acetoacetate decarboxylase only when incubated with the enzyme in the presence of car-bony1 compounds. This resulted in an inhibitory syner- gism between hydrogen cyanide and carbonyl compounds. This synergism was used to investigate the comparative abilities of various carbonyl compounds to form Schiff’s bases at the active site of the enzyme. The order of effec- tiveness among the compounds tested was acetaldehyde > acetone > cyclohexanone > methyl ethyl ketone > 3-hexa- none > diethyl ketone.
2. The ability of hydrogen cyanide to inhibit the enzyme was optimal at pH 6 and decreased abruptly on both sides of this optimum. Although primarily an uncompetitive inhibi- tor of the enzyme, hydrogen cyanide caused perceptible de- creases in the slopes of the lines obtained by plotting kinetic data on reciprocal coordinates. Thus, raising the concen- tration of substrate increased the degree of inhibition caused by a given level of hydrogen cyanide more than could be ex- plained on the basis of combination of the inhibitor only with enzyme-substrate or enzyme-product complexes.
3. Continuous infusion of borohydride into well stirred and well buffered solutions of the enzyme resulted in no loss of activity unless carbonyl compounds, capable of forming Schiff’s base compounds at the active site, were present. The rate of inactivation of the enzyme, under these condi- tions, could be used as an index of the degree of saturation of the active sites by the carbonyl compound being tested and of the reactivity with borohydride of the Schiff’s base compounds so generated at the active site. Quantitative data were obtained in this way for acetone sulfonate, ace- tone, methyl ethyl ketone, and cyclohexanone. Nitrate was observed to protect the enzyme against inactivation by boro- hydride in the presence of acetone.
4. A number of diketones were compared for their abilities to inhibit the enzyme and the structural features essential for this action were discussed. P-Diketones, which inhibited the enzyme, generally did so with perceptible slowness which was accentuated by electron-withdrawing substituents ad-
* The work reported herein was supported in full by Grant GM-10287 from the National Institutes of Health, Bethesda, Maryland.
$ Predoctoral trainee of the National Institutes of Health, Bethesda, Maryland. Present address, Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48104.
$ To whom all requests for reprints should be addressed.
jacent to one of the carbonyl functions. The ability of ace- tylacetone to protect the enzyme against inactivation by borohydride, like its ability to inhibit the enzyme, developed slowly.
5. The reaction of acetoacetate decarboxylase with p-chlo- romercuriphenylsulfonate (CMS) was investigated both in terms of mercaptide formation and in terms of activity. One sulfhydryl group per subunit could be derivatized without loss of activity whereas reaction of a second sulfhydryl was associated with some inactivation. A third equivalent of CMS per subunit reached with an unidentified group to, cause additional inactivation. Amounts of CMS in excess of three per subunit had no further effects and the enzyme retained activity which was impervious to additional CMS. The native enzyme retained 70% of its activity after treat- ment with CMS whereas the fully activated enzyme retained only 50% of its activity after exposure to CMS. Inhibition by CMS was reversed by cysteine.
The mechanism of acetoacetate decarboxylase has been shown to involve the intermediate formation of a Schiff’s base, formed by reaction of the carbonyl group of the substrate with the E-NH2 group of a specific lysine residue (I, 2). Since hydrogen cyanide is known to add across the double bond of azomethines (3-5) it was not surprising to observe that hydrogen cyanide inhibited this enzyme reversibly and with perceptible slowness and that this inhibition by cyanide developed only in the presence of substrate (1, 6). The ability of hydrogen cyanide to protect the enzyme against the substrate-dependent inactivation of the enzyme by borohydride (6) also supported this interpretation of its action. Indeed, several enzymes whose action involves Schiff’s base intermediates have similarly been found to be inhibited by hydrogen cyanide (7, 8). I f hydrogen cyanide does inhibit acetoacetate decarboxylase by forming an adduct with a Schiff’s base intermediate at the active site, then it should aug- ment the inhibition caused by any carbonyl compound capable of forming such an azomethine. This synergism should be useful in detecting the ability of carbonyl compounds to react with the specific E-NH2 group at the active site of acetoacetate decarboxylase. Our conceptions of its action also predict that hydrogen cyanide should be an uncompetitive inhibitor of acetoacetate decarboxylase. Experiments which explore these
Jssuc of October 25, 1970 A. P. L4utol- and I. Fdovich 5215
:Lsl)ects of the interaction of hydrogc~ csyanide with acetoacrtate decarboxylase will be described.
Acrtopyruvate is a potent inhibitor of ncetoacetate drcar- boxylase (9, 10) and protects the enzyme against the substrate- dependent inactivation by borohydridc (6). It has been pro- posed that the interaction of acetopyruvate with this enzyme involves the slow but reversible formation of an enarninc by re- artion with the essential E-NH, group (II). This enamine has been presumed to be formed by tautomerism of a corresponding Schiff’s base compound (II). hcetylacetone is also a potent inhibitor of acetoacetate decarboxylase and its inhibitory prop- erties are similar to those of acetopyruvate (12). It appeared desirable to examine the ability of a number of diketones to inhibit the acetoacetate decarboxylase and to explore the pro- tection afforded by ncetylacetone against the inactivation cauped by borohydride. Such experiments are reported below.
Amino acid analysis of acetoacetate decarboxylase has indi- cated 3 cysteine residues per subunit (13). Titration with Ell- man’s reagent detected only one sulfhydryl group per subunit, while similar titration of the enzyme after denaturation in 4.0 RI guanidinium chloride indicated trT-o sulfhydryl groups per sub- unit. The 3rd residue of cysteine could not be accounted for and the effects of titration of sulfhydryl groups upon the activity of the enzyme were not reported (13). It was therefore of interest to explore the interaction of p-chloromercuriphenyl- sulfonate with acetoacetate decarboxylase in terms of its effects upon the activity of this enzyme. This report also concerns ithrlf with the results of thrse experiments.
MATlCRIALS AND XLTIIODS
Acetoncetate decarboxylase was purified from acetone powders of Clostridium acetobutylicum as described by Zerner et al. (14)
with slight modifications (15). The specific activity of the decarboxylase, used herein, varied from 20 to 323 prior to heat activation (15) and from 40 to 65 after heat activation. This compares favorably with the reported specific activity of the crystalline enzyme (14, 16). Unless otherwise specified, fully activated derarboxylasc was used. Activity was followed in terms of the decrease in absorbance at 270 nm which accorn- panics the decarboxylation oC ncetoacetute (17, 18). Assays
were performed in 0.10 ~1 potassium phosphate buffer at pII 5.9 and at 30”. Lithium acetoacetate KVUS prepared by a modifica- tion (12) of the procedure of Hall (19).
Studies of the ability of various compounds to facilitate the inactivation of the enzymr by borohydride were conducted by exposing the enzyme in 0.10 nr potassium phosphate at pI1 5.9 and at 0” to a rontinuorrs infuLl* 11 of 2.6 ~1 per min of an alkaline solution of sodium borohytlr I&, as previously described (12).
Alcetylacetone was obtained from Fisher. Methylethyl ke- tone, diethyl ketone, and benzoylacetone lvere obtained from Aldrich. Benzenesulfonylncrtorre prepared according to Otto n&l Otto (20) was obtained from Dr. C. R. Hauser. Sodium acetone sulfonate was prepared by a modification (12) of the method of Pa&es and Fisher (2 1). p-Chloromercuriphenyl- sulfonnte was obtained from Sigma. p-Sitrobenxoylacetone was prepared from p-nitroncetophenone by use of the boron trifluo- ride-catalyzrd acetylation with acetic anhydride (22). This material was obtained as pale yellow needles that melted at 11~111.5” (literature 111.4-112.6”). p-Aminobenzoylacetone was prepared from p-nitrobenzoylacetone by reduction with iron in acetic acid (23). This compound was precipitated as the
FIG. 1. Inhibition of acetoacetic decarboxylase by hydrogen cyanide and acetone. Reaction mixtures contained 0.0167 M acetoacet,ate, 0.10 M potassium phosphate, and enzyme in a total volume of 3.0 ml at 21” and pEI5.9. Additional components were: 1 and 5, none; 2 and 6, 0.033 M acetone; 3 and 7, 1.67 X IOV M hvdroeen cyanide: L and 8, 0.033 nr acetone plus 1.67 X 10-O M h;drogen cianide: ‘In the case of Reactions i ---) Q, enzyme was added as the last component after 30 min of equilibration of the other components. In Reactions 5 --f 8, enzyme was present during the 30.min eqnilibrat,ion and acetoacetate was the last component added.
sulfate from et,ha,nol. It was a @-diketone as shown by t’he o-phenSleiledi:Lmiiie t’est (24) and an arylamine as shown by the Bratton and Marshall test (25). -111 other compounds used were obtained from commercial sources at the highest available states of purity.
RESULTS
Inhibition by Hydrogen Cyanide-IIydrogen cyanide had virt,ua,lly no effect on the initial rate of enzymic denarboxylation of a,cetoacetate, but an inhibit’ion did develop during t’he first fern minutes of assay. This inhibition progressed t,o a degree
which was dependent upon the concentrat’ion of hydrogen cya- nide. Preliminary incubation of t.he enzyme or of the substrate wit,h cyanide had no effect on the time course of this inhibit,ion. Because cyanide inhibition developed only in the combined presence of sub&r&e and of enzyme, it seemed possible t,hat cyanide inhibit’ed by virtue of combining &ch one of t,he Schiff’s base compounds which are involved in the catalytic cycle of this enzyme. If t,hia were the case then cyanide inhibition should develop in t,he presence of any compound capable of forming a Schiff’s base with t,he E-KHZ group at the active site of aceto- acetate decarboxylase. The effects of acetone upon the inhibi- tion by hydrogen cyanide were therefore investigated. As illustrated in Fig. 1, 0.033 M a’cetone and 1.67 X lO-‘j M hydrogen cyanide were individually without great effect on the initial rate of the enzymic decarboxylation, whether they were initially incubnt,cd wiiith the enzyme, or not. When cyanide and acetone were simultaneously incubated with the enzyme a marked inhibition of the initial raOe of decarboxylation of acetoacetat,e developed. Thus, acetone did serve to support the gradual development of inhibition by hydrogen cyanide.
FIG. 2. Effects of carbonyl compounds on the inhibition caused by hydrogen cyanide. Reaction mixtures contained 8.35 X 10-Z M
a~etoacetate, 0.10 M potassium phosphate, and 1.4 X 1Oe8 M en- zyme. and the indicated concentrations of HCN in 3.0 ml at nH 5:9 and 25”. Additional components were: 1, none; 9, 0.0233 M
diethyl ketone; 6, 0.0233 M methylethyl ketone; Q, 0.0233 M cyclo-
hexanone; 6, 0.0233 M acetone; 6, 0.0233 M 3-hexanone; and 7, 1.3 X 10d4 M acetaldehyde. Reaction mixtures, excepting the substrate, were equilibrated for 15 min prior to starting the reac- tions by the addition of substrate. Inhibitions are calculated relative to the rate obtained in the absence of both HCN and the carbonyl compounds.
The abilities of several carbonyl compounds to augment the inhibition of acetoacetate decarboxylase by hydrogen cyanide were investigated. This was done by incubating the enzyme with several levels of cyanide and of each of the carbonyl com- pounds for 15 min at 25” before adding substrate and recording the initial rate of decarboxylation. Fig. 2 presents the results of these studies. Two separate phenomena are illustrated in Fig. 2. The first of these is the ability of carbonyl compounds them- selves to inhibit the decarboxylase, while the second is their ability to enhance the inhibition because of hydrogen cyanide. The first would relate to the ability of these compounds to bind to the catalytic site, whereas the second effect would depend upon their ability, once on that site, to form an azomethine capable of reacting with hydrogen cyanide. Thus diethyl ketone (Line 2) inhibited the decarboxylnse rather well but scarcely augmented the inhibition by hydrogen cyanide. Methylethyl ketone (Line S) did not inhibit as well as diethyl ketone but was more effective in augmenting the inhibition by hydrogen cyanide. Acetone (Line 5) which inhibited the enzyme about as well as diethyl ketone, was a much more effective synergist of hydrogen cyanide inhibition. Considering its low concentration (1.3 x low4 M) acetaldehyde (Line 7) was very effective both as an
inhibitor and as a synergist of hydrogen ryanide. Acetaldehyde at 0.0233 M inhibited the decarboxylase 82%. From the manner of presentation of the data in Fig. 2 it would appear that 3.hexa- none did not synergize with hydrogen cyanide. This appear- ance is a consequence of the high degree of inhibition caused by 3-hexanone itself and by the necessary limit of inhibition at 100%. If the activity in the absence of hydrogen cyanide was in each case taken to be an uninhibited rate, then the inhibitions calculated as a function of the concentration of hydrogen cyanide would clearly expose synergism. This is shown in Fig. 3.
FIG. 3. Synergism between carbonyl compounds and hydrogen cyanide. The data shown in Fig. 2 are here presented in a manner designed to more clearly expose synergistic effects. Inhibitions are calculated relative to the rates obtained in the absence of HCN.
If we consider that hydrogen cyanide reacts reversibly with an enzyme-substrate or enzyme-product complex to yield an inac- tive adduct then the association constant may be written as
K [ES - HCN] (Vo - Vi)
acN = IESllHCNl = (Vi) (HCN)
where Vo is the activity in the absence of cyanide, Vi is the linear inhibited rate achieved in the presence of cyanide and (HCN) is the concentration of hydrogen cyanide. This association con- stant for cyanide was investigated as a function of pH and the results are illustrated in Fig. 4. Hydrogen cyanide was opti- mally effective as an inhibitor at pH 6.0 and its potency de- creased abruptly on both sides of that optimum. In calculating these values of R noN it was necessary to take as Vi the linear rates achieved after the inhibition by a given level of hydrogen cyanide had reached its equilibrium. Achievement of this state of inhibition equilibrium was hastened by working at 30” and at relatively high concentration of acetoacetate. The amount of substrate consumed during the time needed to reach inhibition equilibrium was insignificant relative to the total concentration of substrate.
If hydrogen cyanide inhibits the decarboxylase only by com- bining with enzyme-substrate or an enzyme-product complexes then in a Lineweaver and Burk (26) analysis it should behave as an uncompet,it,ive inhibitor. This was explored and the results are shown in Fig. 5. Once again, these data represent rates taken after inhibition equilibrium had been reached. Low concentrations of enzyme and a correspondingly compressed time scale was used to make certain that subst’rate consumption was negligible during the few minutes required to achieve inhibi- tion equilibrium. The results in Fig. 5 reveal that hydrogen cyanide was more than merely uncompetitive with respect to acetoacetate. It actually decreased the slope of the Lineweaver and Burk lines. This could be accounted for if hydrogen cyanide were able to inhibit by some mechanism in addition t,o combining with enzyme-substrate or enzyme-product complexes.
E$ects of Borohydride-The observations that acetone acts as an inhibitor of acetoacetate decarboxylase (2, 9) and that the
Issue of Odober 25, 1970 A. P. Autor and I. Fridovich
EFFECT OF pH ON CYANIDE INHIEIITION
5217
50 6.0 7.0 PH
FIG. 4. Effect of pH on inhibition by hydrogen cyanide. The association constant for the react.ion between HCN and the en- zyme, in the presence of substrate, is here given as a function of pH. Reaction mixtures contained 0.0275 M acetoacetate, 0.10 M potassium phosphate, 0.10 M potassium citrate, enzyme, and 1.3 X 10-e M HCN in 2.2 ml at 30” and at the indicated pH. The basis for the calculation of the association constant Kc,,- is described in the t,ext. In this region of the pH scale virtually all of the cyanide was present as HCN and the association constant, although pre- sented on the ordinate as Kc,- , refers to tot’al cyanide.
EOO-
lOOO[
I I I I I I I I 0 40 80 120 160 200 240 280 320
‘/Ace+oacetote
FIG. 5. Lineweaver and Burk analysis of inhibition by hydro- gen cyanide. Reaction mixtures contained enzyme, 0.10 M potas- sium phosphate, the indicated concentrations of acetoacetate, and the following concentrat’ions of hydrogen cyanide: Line I, none; Line d, 0.67 X 10-G M; Line 3, 1.33 X lo-” M. The reaction volume was 3.0 ml and reaction conditions were pH 5.9 and 30”. The points represent rates taken after inhibition equilibrium had been achieved.
enzyme catalyzed an exchange of deuterium between acetone and water (2) indicated the likelihood that acetone forms a Schiff’s base with the essential E-NH2 group. The ability of acetone to augment the inhibition by hydrogen cyanide strength- ens this conclusion. One would then anticipate that acetone should facilitate the inactivation of the enzyme by borohydride. Given a constant level of borohydride, maintained by the balance
FIG. 6. The effect of acetone on inactivation of the enzyme by borohvdride. Potassium borohvdride. 0.002 M in 0.10 N NaOH. was&used at a rate of 2.6 ,nl per min into 0.9 ml of magnetically stirred reaction mixture containing enzyme and the indicated concentrations of acetone in 0.10 M potassium phosphate at pH 5.9 and 0”. Aliquots of the reaction mixture were removed at intervals for assay of residual decarboxylase. The rate of inac- tivation was first order and pseudo first order rate constants are here presented as a function of (acetone) on reciprocal coordinates.
100 1
,,t/ I r I I I I I I I I
0 20 40 60, 80 100 120 140 160
“Cyclohexanone
FIG. 7. Effect of cyclohexanone on inactivation of the enzyme by borohydride. Conditions are as described in the legend of Fig. 6 except that cyclohexanone was used in place of acetone.
between a continuous infusion (12) and the proton-dependent hydrolysis (27), one would expect that the rate of inactivation of the enzyme should reflect the degree of saturation of the sensi- tive sites by acetone. It should thus be possible to arrive at a dissociation constant for acetone in terms of its ability to support the inactivation of the enzyme by borohydride. Similar meth- ods have been used to arrive at a dissociation constant for 2-0x0- propane sulfonate which was identical with the Kr obtained from studies of the competitive inhibition of the enzyme by this substrate analogue (12). The results of a study of the acetone facilitated inactivation of the enzyme by borohydride are pre- sented in Fig. 6. Potassium borohydride, 0.002 M in 0.10 N XaOH, was infused at a rate of 2.6 ~1 per min into 0.9 ml of a stirred reaction mixture containing enzyme and the indicated concentrations of acetone in 0.10 M potassium phosphate at pH 5.9 and at 0”. Aliquots (50 ~1) of this reaction mixture were removed at intervals and assayed at 30”. The rate of inactiva- tion of the enzyme was a first order dependent of the residual active enzyme and the rates of inactivation by borohydride at any given level of acetone could conveniently be expressed in terms of a pseudo first order rate constant. In Fig. 6 these rate constants are plotted as a function of the concentration of acetone on reciprocal coordinates and from the resultant line K, was found to be 0.20 M and I’,., to be 0.625 mini. Control experi- ments in which the enzyme was exposed to an infusion of boro- hydride in the absence of acetone or to acetone in the absence of borohydride caused no inactivation. Fig. 7 presents the
TABLE I Facilitation of inhibition with potassium borohydride at 0’
The effects of carbonyl compounds upon inactivation of aceto- acetate decarboxylase by a continuous infusion of borohydride. Data, such as that represented by the points in Figs. 6 and 7 were used to define straight lines when plotted on reciprocal coordinates and from the slopes and intercepts of such lines values of K, and V,,, were obtained. In all cases the conditions used were those described for the specific cases of acetone and cyclohexanone in Figs. 6 and 7.
TABLE II Effect of nitrate and ðyl ketone on rate of acetone-facilitated
borohydride inactivation
Effects of nitrate and of diethyl ket~one on the acetone-sup- ported inactivation of acet.oacetate decarboxylase by a continuous infusion of borohydride. Potassium borohydride was infused int,o 0.9 ml of a solution containing enzyme, 0.22 M acetone, 0.10 M
potassium phosphate, and the indicated additions, at 0” and pH 5.9. At intervals, aliquots were removed, diluted and assayed for decarboxylase activity. The rate of loss of activity was in each case characterized by a pseudo first order rate constant.
Acetone Nitrate Diethyl ketone ’ k
M M .w min-’
0.223 0.342 0.223 1.1 X 10-a 0.051 0.223 2.8 x 10-a 0.027 0.223 0.278 0.223
Protection .I.
%
85 92 35
-
results of a similar experiment in which the ability of cyclo- hexanone to support the inactivation by borohydride was ex- plored. Acetone sulfonate, methylethyl ketone, and diethyl ketone were also investigated in this way and the kinetic con- stants so derived are presented in Table I.
E$ects of Nitrate and of Diethyl Ketone on Inactivation by Borohydride-Acetoacetate decarboxylase is reversibly inhibited by monovalent anions. The effects of pH and the thermody- namic parameters of the inhibitions caused by a series of mono- valent anions were compatible with the proposal that binding of inhibiting anions to the enzyme involved pairing of the anions with a cationic acid group and migration of the resultant ion pair from the aqueous phase into a predominantly hydrophobic environment (17). It has been proposed that the great efficiency of borohydride as a reductant of azomethines generated at the active sit.e of the enzyme depends, in part, upon the binding of borohydride to the active site, as an anion, prior to its action as a reductant (12). The ability of nitrate to protect against the acetone sulfonate-facilitated inactivation by borohydride was taken as support for this concept (12). Since acetone sulfonate is an anion, one could, however, argue that the protective effect of nitrate was exerted by hindering the binding of acetone sulfo- nate, rather than that of borohydride. This argument could not
FIG. 8. Inhibition of acetoacetate decarboxylase by benzoyl- acetone. Reaction mixtures contained 0.0167 M acetoacetate, enzyme, 0.10 M potassium phosphate, and the following concen- trations of benzoylacetone: 1, none; 2, 1.67 X lo+’ M: d, 3.33 X lo+ NI; 4, 6.67 X lob5 M, in a total volume of 3.0 ml at pH 5.9 and 30”. Enzyme was the last component added.
be raised with respect to an uncharged molecule such as acetone- The ability of nitrate to protect against the acetone facilits.ted inactivation by borohydride was therefore investigated. The results are presented in Table II. It is apparent that even low concentrations of nitrate exerted a strong protective effect.
Diethyl ketone, 0.22 M, did not support the inactivation of acetoacetic decarboxylase by borohydride (Table I), but as shown in Table II it did weakly protect against the acetone facilitated inhibition by borohydride.
Inhibition by Diketones-Both acetopyruvate (10, 11) and acetylacetone (l2)l inhibit the enzyme powerfully and with perceptible slowness and it appeared probable that other @- diketones would do likewise. Fig. 8 presents the effects of benzoylacetone on the activity of the enzyme. It is apparent that benzoylacetone inhibits strongly and that this inhibition develops with perceptible slowness. The linear rates achieved after approximately 4 min did represent a state of inhibition equilibrium. This was indicated by repeating the experiment whose results are shown in Fig. 8 but changing the order of addition of reactants so that the enzyme was incubated with benzoylacetone for 15 min prior to the addition of substrate. When this was done the initial rates were zero but increased within a few minutes to linear rates identical with those shown in Fig. 8. Kinetic analysis of the inhibition by benzoylacetone indicated that its action was largely that of a competitor with respect to acetoacetate. This is shown in Fig. 9. There is a small intercept effect which indicates that benxoylacetone had some ability to combine even with enzyme-substrate or enzyme- product complexes. From these data, K, for acetoacetate was found to be 7 X lop3 M while KI for benzoylacetone was 1.9 X lo-” M. The points in Fig. 9 represent linear rates and were taken after inhibition equilibrium had been achieved. A diluted enzyme and a correspondingly compressed time scale permitted these linear rates to be reached before significant depletion of the substrate had occurred.
A variety of diketones were tested for their ability to inhibit acetoacetic decarboxylase after allowing a 15-min incubation to
1 The dissociation constants reported in this reference for the reaction of acetylacetone with acetoacetate decarboxylase were correct, but the association constant of 7 X lo7 M represents a typographical error and should have been 7 X lo5 M.
Issue of October 25, 1970 A. P. Autor and I. Fridovich 5219
300 -
260-
220-
0 40 60 120 160 200 240 260 320
FIG. 9. Lineweaver and Burk analysis of inhibition by benzoyl- acetone. Reaction mixtures contained 0.10 M potassium phos- phate, enzyme, the indicated concentrations of acetoacetate and the following amounts of benzoylacetone: 1, none; 2,3.33 X 10e6 M;
3,6.67 X 1OF M in a total volume of 3.0 ml at 30” and pH 5.9. The points represent rates taken after inhibition-equilibrium had been achieved.
TABLE III
Effect of time on protection by acetyl acetone
Effect of time on the ability of acetylacetone to prot’ect aceto- acetate decarboxylase against inactivation by borohydride. Acetylacetone was added to a final concentration of 2 X 10e4 M
to magnetically stirred reaction mixtures containing enzyme, 0.020 M acetone sulfonate, and 0.10 M potassium phosphate at 0” and pH 5.9. Total reaction volume was 0.5 ml. Aft.er a specified interval, 10 ~1 of 0.005 M borohydride in 0.005 M NaOH was added and 50 pl of the reaction mixture was subsequently taken for assay of decarboxylase activity.
3.3 x 10-3. 0 0.00594 0 3.3 x 10-5, 1.0 0.0067 6.5 3.3 x 10-b. 8.0 0.0129 57.0 3.3 x 1O-5. 16 0.0148 72.5 No BH,-. 0.0182
accommodate possible slowly developing inhibitions. The results indicated that /3-diketones were strongly inhibitory but that other diketones were not effective. Thus, 3.3 X 1OP M acetyl- acetone inhibited 94% whereas 1.7 X 1OP M acetonylacetone did not inhibit at all. Thus, 1 X 10e4 M 1,3-cyclohexanedione inhibited 50% but the 1,2- and the 1,4-cyclohexanediones did not inhibit at all. Within the class of P-diketones, bulky sub- stituents on the methylene group o( to one of the carbonyls or on the methylene group between the carbonyls had only modest effect but bulky substituents on the methylene groups adjacent to both carbonyls eliminated inhibitory activity. Thus 6.7 X lo-5 M benzoylacetone inhibited 92%, and 1.7 X 1OP M 3-ben- xylidine-2,4-pentanedione inhibited 43yo whereas 6.7 X low5 M
dibenzoylmethane did not inhibit at all. The rate of inhibition by a particular /3-diketone seemed to relate to the electron-with- drawing effect of the substituent on the methylene group a to one of the carbonyls. Thus 1 X low4 M p-aminobenzoylacetone inhibited 65% whether previously incubated with the enzyme or not, whereas 1 X 10-4 M p-nitrobenzoylacetone inhibited 88%
TABLE IV Molar extinction coeficients for mercaptide formation
by CMS
Molar extinction coefficients for mercaptide formation by CMS. Aliquots (10 ~1) of 3 X lo+ M cysteine hydrochloride were added to 3.0 ml of 5 X lo-$ M CMS in 0.10 M potassium phosphate at 25” and at pH 5.9. Absorption spectra were recorded at intervals against a blank lacking only the CMS. The molar extinction coefficients here tabulated were calculated from the family of spectra thus generated.
when incubated with the enzyme for 30 min at 30”, 57% when incubated with the enzyme for 10 min and 10% when no time was allowed for incubation with the enzyme. Benzenesulfonyl acetone inhibited strongly but very slowly. Thus 6.7 X 10m5 M
benzenesulfonyl acetone inhibited 57 y0 at. inhibition equilibrium. At 30”, 10 min was the time required for half of this equilibrium inhibition to be achieved. Benzene sulfonylacetone is not, strictly speaking, a P-diketone but the structural analogy is clear and the electron-withdrawing capacity of the sulfonyl group probably accounts for the slow rate at which it inhibited the enzyme.
Acetylacetone protected the enzyme against the acetone sulfonate-supported inactivation by borohydride (12). Since inhibition of the enzyme by acetylacetone developed with per- ceptible slowness it was of interest to determine whether its ability to protect against the acetone sulfonate-supported inac- tivation by borohydride also developed slowly. Enzyme was added to 0.5 ml of magnetically stirred reaction mixtures con- taining 0.020 M sodium acetone sulfonate, 2 X low4 M acetyl- acetone, and 0.10 M potassium phosphate at pH 5.9 and at 0”. After a specified time, 10 ~1 of 0.005 M sodium borohydride in 0.005 N NaOH was added and a 50+1 aliquot was subsequently taken for assay of decarboxylase activity. The results shown in Table III show that the ability of acetylacetone to prot.ect the enzyme against inactivation by borohydride, like its ability to inhibit the enzyme, develops slowly. When the sodium boro- hydride was added to such reaction mixtures 15 set prior to the addition of enzyme, no inactivation was observed; in accord with the rapid acid-catalyzed hydrolysis of borohydride (27).
Effects of p-Chloromercuriphenylsulfonate-The quantitative aspects of the interaction of CMiY and cysteine were investigated by spectrophotometric titration. Only one CMS was found to react per cysteine and the changes in molar extinction which accompanied this reaction are listed in Table IV as a function of wave length. A slight excess of CMS was added to acetoacetate decarboxylase (3.6 CMS per subunit) and the rate of mercaptide formation was followed at 235 nm. As shown in Fig. 10, ap- proximately two sulfhydryls reacted rapidly and there was sub- sequently a very slow increase in absorbance at 235 nm. The
Z The abbreviation used is: CMS, p-chloromercuriphenylsulfo- nate.
FIG. 10. Rate of reaction of CMS with acetoacetate decarboxyl- ase. At time zero, 3.6 equivalents of CMS per subunit of enzyme were added to reaction mixtures at 25” and buffered at pH 5.9 by 0.10 M potassium phosphate. Reaction was recorded in terms of increase in absorbance at 235 nm. Line 1 represents the reaction of native enzyme whose specific activity was 28 units per mg. Line B was obtained with heat-activated enzyme whose specific activity was 65 units per mg.
40 1
20 k
L 2,, , , ,
0 10 20 30 40 50 60 Minutes at 25”
FIG. 11. Rate of inhibition of acetoacetate decarboxylase by CMS. 3.6 equivalents of CMS were added per subunit of enzyme as described in the legend of Fig. 10. At intervals thereafter, aliquots of the reaction mixture were diluted 125-fold with buffer (Line I) or with 1 X ~O-*M cysteine in buffer (Line 2) and after a delay of 30 min these aliquots were assayed for decarboxylase ac- tivity. A illustrates the results obtained with fully activated enzyme whereas B presents the behavior of native enzyme.
significance of the slight difference seen between the native and the fully activated enzymes is not clear.
This experiment was repeated but this time the effects of CMS (3.6 per subunit) on the activity of the enzyme was moni- tored by removing aliquots as a function of time and diluting them 125-fold with buffer or with 1 X 10” M cysteine in buffer, prior to assay. Cysteine, per se, had no effect on the assay of the enzyme. Fig. 11 illustrates the results of these manipula- tions. The inhibition by CMS exhibited two phases, the first rapid and the second very much slower. These rates of inactiva- tion paralleled the rates of mercaptide formation shown in Fig. 10. The rapid phase of inhibition by CMS ceased with the loss of 50% of the activity in the case of the fully activated enzyme and of only 30% of the activity in the case of the native enzyme. In
2.5
2.0 ‘2 3
c 1.5 z !- YE 1.0 E
o.5g
4
FIG. 12. Titration of acetoacetate decarboxylase with CMS. Increments of CMS were added to native decarboxylase (Line 1) and to fully activated decarboxylase (Line 2) in 0.10 Y potassium phosphate at 25” and pH 5.9. Mercaptide formation was moni- tored at 235 nm and aliquots were simultaneously removed for assay of decarboxylase activity. A presents the effects of titra- tion with CMS upon the activity while B illustrates mercaptide formation with the heat-activated enzyme under the same condi- tions.
the case of the fully activated enzyme, the rapid phase of inhibi- tion was reversed by cysteine whereas the slow phase was not. In the case of the native enzyme all of the inhibition appeared to be reversed by cysteine but the process was not followed long enough to exclude the possibility of a slow and irreversible inactivation.
The enzyme was then titrated with CMS. Activity and mercaptide formation were measured and the results are pre- sented in Fig. 12 as a function of the amount of CMS added. It is apparent that the first equivalent of CMS, although involved in mercaptide formation, caused virtually no loss of activity. In contrast, the second equivalent of CMS, which was also in- volved in mercaptide formation, did cause loss of activity. The third equivalent of CMS, which gave no spectrophotometric indication of having reacted with the enzyme, caused additional inactivation. The activity lost by the native enzyme is again seen to be less than that lost by fully activated enzyme. The plateau of mercaptide formation shown in Fig. 12B is 20% greater than would have been expected on the basis of strict stoichiometry, the reason for this is not clear but may relate to the fact that nearly 90 min elapsed during the titration of a given sample of enzyme. Slow and irreversible changes in the protein-CMS compound may have contributed to changes in absorbance at 235 nm, upon which basis mercaptide formation was calculated.
DISCUSSION
Hydrogen cyanide appears to inhibit the decarboxylase by combining with Schiff’s base compounds formed at the active site. Were this its sole mode of inhibition, one would anticipate that it would exhibit the properties of a classical uncompetitive inhibitor; that is, an inhibitor which acts by combining with enzyme-substrate or enzyme-product complexes but which cannot act upon the free enzyme and which therefore has no effect on the slopes of Lineweaver and Burk lines. Fig. 5 shows that hydrogen cyanide decreased the slopes of such lines. One explanation which may be advanced to explain this effect is that a cyanohydrin is formed by reaction of the substrate and the inhibitor and that this cyanohydrin is a more potent inhibitor of
Issue of October 25, 1970 -4. P. Autos- and I. Fridovich 5221
the decarboxylase than is hydrogen cyanide itself. This hypoth- esis does not exhaust the possibilities but no experiments were done to clarify this point, so that further discussion is not war- ranted.
The ability of carbonyl compounds to augment the inhibition by hydrogen cyanide and to do so only when incubated with en- zyme is indicat,ive of their ability to form Schiff’s bases at t’he actire site. Their ability to support the irreversible inactiva- tion of t#he enzyme by borohydride is a measure of the same cnpa- bility. The striking differences between acetone, methylethyl ketone, and diethyl ketone indicat.es that there are stringent limitations on the availability of the E-NH2 group at the active sit,e for Schiff’s base formation with added carbonyl compounds. Differences between abilities to inhibit the enzyme and to syner- gize 17.ith hydrogen cyanide indicate that binding of a carbonyl compound onto the enzyme is not synonymous with the forma- tion of a Schiff’s base. The variation of the inhibitory pot’ency of hydrogen cyanide with pH parallels the variation of the cataly- tic efficiency of the enzyme with PH. This suggests that forma- tion of Schiff’s base intermediates is the rate-limiting portion of the catalytic process.
The abilit,y of carbonyl compounds to support irreversible in- activation of the enzyme by borohydride is a reliable indicator of their tendency to form Schiff’s base compounds at the active site. The technique of maintaining a co&ant concentration of borohydride by cont,inuous infusion and of then measuring the effects of the concentration of the carbonyl compound on the rate of inactivat.ion provided quantitat,ive data. The dat,a so derived reflect both the tendency of a particular carbonyl com- pound t,o form a Schiff’s base at the active site (Ii,), and the reactivity of the Schiff’s base so formed with borohydride (V,,,,) . Table I shows t’hat acetone sulfonate had a K, which was two orders of magnitude lower than that of acetone. This is in ac- cord with the existence of a 2nd lysine residue adjacent to the active site lysine (28) and with the evidence for a positively charged milieu at the active site (29). Thus, electrostatic inter- actions would have the effect of attracting the anionic acetone sulfonate to the active site and of thus increasing its effective concentration in the vicinity of that, site, whereas no such effect would be operative in the case of acetone. On the other hand, the V,,, for acetone V&S 5 t,imes higher than that for acetone sulfonate. In explanation of this we might invoke the electro- static interactions of borohydride with the susceptible Schiff’s base. -2 nearby negative charge, such as that on the sulfonate group, should diminish the affinity of the borohydride anion for the reducible Schiff’s base. For met,hylethyl ketone and cyclo- hexanone, which gave V,,, values lower than that of acetone, Iye call propose steric problems, which apparently become so serious in the case of diethyl ketone that, 110 ability to support borolrydride inactivation was evident.
The ability of nitrate t,o protect against the acetone-supported iinactivation by borohydride supports the proposal (12) that borohydride is bound to the susceptible site as a monovalent anion prior to its action as a reductnnt. It follows that inhibi- tory monovalent anions bind to the active site. Since previous st.udies of the inhibition of acetoacetic decarboxylase by anions (17) suggested that binding to the sensitive site involved a migra- tion of the inhibitory anions from water into a hydrophobic en- vironment, we may conclude that the active site of this enzyme
is in or is at least very close to a hydrophobic cluster. Its ability
to discriminate between met.hylethyl ketone and diethyl ketone further suggests that the act,ive site is in a rather small crevice.
@-Diketones were potent inhibitors of the decarboxylase and their inhibitions were typically slow in developing, although p-aminobenzoylacetone did inhibit rapidly. The abilit,y of ,&di- ketones to protect the enzyme against inactivation by boro- hydride, like their abilit,y to inhibit the activity of the enzyme, was slow to develop. This suggests that both of these effects of fi-diketones reflect their association with the same site on the enzyme and that is the active site. I f @diketones combine with the active site and so prevent other compounds from forming Schiff’s bases at t,hat site, as they must to protect against boro- hydride, then P-diketones should be competitive inhibitors of the enzyme. The data for acetylacetone (12) and for benzoylace- tone indicated a small but. definite noncompetitive effect,, in addi- tion to a larger competitive effect. Thus, fl-diketones appear able to combine with enzyme-substrate or enzyme-product com- plexes to some extent,. In the case of acetylacetone, the non- competitive component of its inhibition disappeared as the tem- perature was raised, suggesting that combination with enzyme complexes was based upon weaker bonds than was combination with the free enzyme. The chemical basis for the interaction of /3-diketones with the active site of the free enzyme probably in- volves the formation of an enamine. Earlier studies of the inhibition by acetylacetone (12) and of acetopyruvate (11) have led to the proposal of an enamine adduct. It was not possible to decide, on the basis of these studies (II), which of the carbonyl groups of acetopyruvate reacted with the E-amino group at the active site of the enzyme. The results now obtained with a series of /%diketones implicates t’he carbonyl group adjacent to the methyl group. Thus, the st,ructure of inhibit,ory P-diketones could be generalized as:
Since the ,&carbonyl group could be replaced by ot.her electron- withdrawing functions, such as a sulfonyl group, it cannot be the group direct.ly involved in reaction with the enzyme’s essential E-NH2 group. The carbongl adjacent to the methyl group must then, be the reactive group. If the formation of enamines be- tween inhibitory fi-diketones and the active site did involve prior formation of a Schiff’s base then @-diketones might be expected to support some inactivation by borohydride. They did not do so to any detectable extent whether they had been incubated with the enzyme before the infusion of borohydride was begun of whether they were added to the reaction mixture after the in- fusion of borohydride was under way. Some route of enamine formation not involving a Schiff’s base intermediate is indicated. The enamine could, for example, be generated directly by de- hydration of the carbinolamine adduct formed by reaction of the P-diketone with the essential E-NH, group.
Each subunit of acetoacetate decarboxylase contains two sulf- hydryl groups which react rapidly with CYIS. The first of these, in terms of its avidity for CMS, is apparently not essential for the catalytic integrity of the enzyme, since its titration with CMS caused no loss of activity. When a second equivalent of CMS was added per subunit it reacted with the second sulfhydryl group and caused a partial loss of activity. The third equivalent
of CMS apparently reacted with other than a sulfhydryl group since it caused some loss of activity but gave no spectrophoto- metric indication of mercaptide formation. The loss of activity caused by CMS was of two types. The rapid inactivation was reversed by cysteine whereas the much slower inactivation was not reversible and probably reflected a slow denaturation of the CMS-derivatized enzyme. It is very interesting that the native enzyme lost approximately one-quarter of its activity during the rapid reaction with CMS whereas the heat-activated enzyme lost one-half of its activity under the same conditions. Besides the obvious difference of specific activity, this is the only difference yet detected between these forms of the enzyme. This suggests that the process of heat activation involves minor changes in conformation, one of the sequelae of which is a greater sensitivity to the derivatization of its sulfhydryl groups.
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