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Pergamon 0020-71 lX(93)EOOlO-MIn/. J. Bio&m. Vol. 26, No. 3. pp. 309-3 IX, I994Copyrig ht B; 1994 Elsevier Science LtdPrintedn Greal Britain. All rights reserved0020.7 I IX/94 6.00 + 0.00

MINIREVIEWTHE ACTIVE SITE AND MECHANISM OF THE

/I-GALACTOSIDASE FROM ESCHERICHIA COLIR. E. HUBER, M. N. GUPTA~ and S. K. KHARE*

Division of Biochemistry, Faculty of Science, University of Calgary, Calgary, Alberta, Canada T2N 1NR[Fax (403) 289-931 I] and *Department of Chemistry, Indian Institute of Technology, Hauz Khas,

New Delhi 110016, India

(Received 12 August 1993)

INTRODUCTIONfl-Galactosidase of Escherichia coli is an idealmodel for study of enzymes that act on disac-charides. It is stable and easy to isolate in largequantities and it is easy to carry out site specificsubstitutions with this E. col i enzyme. Electronmicrographs (Karlsson et al., 1964) indicatedthat the enzyme is tetrameric with monomers atthe corners of a 120 A square that is 70 A high.Recent preliminary X-ray studies (Jacobson andMatthews, 1992) have confirmed this. The en-zyme has four binding sites (Cohn, 1957).Fowler and Zabin determined the amino acidsequence of j3 -galactosidase (Fowler, 1972,1978a,b; Fowler and Zabin, 1970, 1978a,b,c;Fowler et al., 1978a,b; Langley et al., 1975;Zabin and Fowler, 1972) and Kalnins et al.,(1983), the DNA sequence. Each identicalmonomer has 1023 amino acids and a molecularweight of 116,353. The enzyme is composed of35% u-helix, 40% P-sheet (mainly parallel),12% random coil, and 13% /?-turn (Arrondoet al., 1989).

ACTIVE SITE COMPONENTS1) Tyr

p-Galactosidase activity is partly controlledby a group that loses a proton in the alkalinerange (Tenu et al., 1971; Withers et al., 1978). Astudy of the binding of positively charged in-Abbreviarions: MNPG, m-nitrophenyl-p-galactopyrano-

side; ONPG, o-nitro-phenyl-/7-o-galactopyranoside;PNPG, p-nitrophenyl-/I-D-galactopyranoside; mNP, M-nitrophenol: oNP, o-nitrophenol; pNP, p-nitrophenol.

hibitors and corresponding neutral inhibitorsindicated that there is a group at the active sidewith a pKa of 9.4 in the presence of neutralinhibitors and a pKa of 8.0 with positivelycharged inhibitors (Loeffler et al., 1979). Theauthors reasoned that a Tyr is at the active site./3-Galactosidase with >80% of the Tyrsubstituted by m-fluorotyrosine (Ring et al.,1985) has pH properties that can be predictedon the basis of the different pKas of Tyr andm-fluorotyrosine.

Lactoperoxidase causes /?-galactosidase tobecome inactivated and Tyr-253 becomes heav-ily iodinated (Huber et al., 1982). IPTG (acompetitive inhibitor) decreases both inacti-vation and iodination. Several other Tyr alsobecome iodinated to lesser extents (Edwardsand Huber, 1986) and, since the inactivationwas stoichiometric with iodination, it is prob-able that the activity loss is an effect of thecombined iodination of any of the Tyr. If/I-galactosidase is iodinated, it becomes highlysusceptible to chymotrypsin proteolysis (Ringand Huber, 1993). The inactivation effect ison the catalytic action of the enzyme not thebinding (Ring and Huber, 1993).

Tyr-588 may be important. Chymotrypsinaction on native P-galactosidase cleaves thepeptide bond beside Trp-585 and this causes atotal loss of activity (Edwards et al ., 1988). Therate at which this bond is cleaved is highlydependent upon what ligands are at the activesite. Inhibitors, bivalent metals and monovalentmetals all modulate the inactivation. Tyr-588 isonly 3 residues removed from the bond that iscleaved and the Tyr at that position is conserved

309

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310 R E HUBER et alin almost all species studied. Recent studies(Huber, unpublished) have shown, however,that when Tyr-588 is replaced by Phe, theenzyme is still active, albeit very susceptible tointracellular proteolysis.

Studies with an active site directed inhibitorthat reacts with Met 502 (Sinnott and Smith,1976, 1978; Fowler et al., 1978~) led to thesuggestion that Tyr-503 is important. Met-502itself is not important since a /?-glactosidaseisolated from a Met-mutant of E. col i grownon norleucine-that replaced Met-was active(Naider et al., 1972). A Tyr equivalent to thatat position 503 is conserved in every /?-galactosi-dase for which a sequence has been determined(Stokes et al., 1985; Buvinger and Riley, 1985;Schmidt et al., 1989; Schroeder et al., 1991;Hancock et al., 1991; Poch et al., 1992; Burch-hardt and Bahl, 1991; David et al., 1992) andsite specific replacements (Ring et al., 1988;Cupples and Miller, 1988; Ring and Huber1990) for Tyr-503 cause the activity to be low-ered about 2000-fold. When a His was substi-tuted, the pH profile was altered as expected foran enzyme with a His acting as a base catalyst.The activity of the Phe substituted enzyme withONPG was reduced about 2000-fold while therate with a pyridinium salt substrate (whichcannot be acid catalyzed) was only reducedabout IO-fold. Thus, acid/base catalysis by Tyr-503 may provide a rate increase of about 200-fold. This is not a large rate increase but itwould be beneficial to a cell because 200Xless enzyme would have to be synthesized forequivalent activity.(2) Glu

Tenu et al. (1971), suggested that a carboxylgroup might be at the active site. Studies byHerrchen and Legler, (1984) showed that Glu-461 was modified by an active site directedinhibitor, conduritol C cis-epoxide. An analysisof sequences (Stokes et al., 1985; Buvinger andRiley, 1985; Schmidt et al., 1989; Schroederet al., 1991; Hancock et al., 1991; Poch et al.,1992; Burchhardt and Bahl, 1991; David et al.,1992), shows that a Glu equivalent to Glu-461is strictly conserved. Further evidence of theimportance of Glu-461 has come from site-specific replacements (Bader et al., 1988; Cup-ples and Miller, 1988; Cupples et al., 1990) thatresult in large decreases in the activity. Inaddition, Mg+ binding is dramatically de-creased by substitutions for Glu-461 which donot have a negative charge (Edwards et al.,

1990). Glu-461 is, thus, probably a Mg2+ ligand.Substitution of Glu-461 by a neutral or a posi-tively charged residue also causes a loss of thebinding of positively charged transition stateanalogs. Glu-461 may thus be involved in elec-trostatic stabilization of a galactosyl cationtransition state.

There is now some good evidence suggestingthat Glu-461 may not be the residue involved inthe formation of a covalent bond to galactose.Gebler et al. (1992) showed that 2-deoxy-2-fluoro-/3-D-galactopyranoside becomes cova-lently attached to Glu-537 when B-galactosidaseis incubated with 2,4-dinitrophenyl-2-deoxy-2-fluoro-/?-D-galactopyranoside. Yuan et al.(1994) have now confirmed (by site specificreplacement) that Glu-537 is important forcatalysis.(3) Metal cofactors

Monovalent cations (Na+ and K+) activatej?-galactosidase (Lederberg, 1950; Wallenfelsand Weil, 1972; Neville and Ling, 1967; Cohnand Monod, 195 1; Becker and Evans, 1969;Strom et al., 1971; Hill and Huber, 1971, 1974)but the specific role that the monovalent cationsplay is not clear. Divalent metal ions (Mg*+ orMn*+) are also important (Case et al., 1973;Huber et al., 1979). One divalent metal bindsper monomer with a dissociation constant ofabout 1 PM (Tenu et al., 1972; Huber et al.,1979). Magnetic resonance has located Mn*+ towithin 8-9 A of the aglycone part of a substrateanalog inhibitor bound to the active site(Loeffler et al., 1979) and a large shift of thealkaline inflection point of the pH profile (from6.5 to 8.4) occurs upon binding Mg+ (Tenuet al., 1971). One of the ligands for Mg*+binding may be Glu-461 (Edwards et al., 1990).Again, the exact role that Mg*+ or Mn2+ playsis not yet known.

BINDINGSPECIFICITY(1) Galactose sub -site

Sugars with differences in hydroxyl grouporientation (from those of D-galacose) at pos-itions 3 or 4 do not bind to free enzyme (Huberand Gaunt, 1983) and, changes at position 6decrease the binding. Brockhaus et al. (1979)and Huber et al. (1986) showed that substrateswithout a hydroxyl at position 2 bind readily,but do not react. Thus, the 2 position may be ofsome importance.

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j?-Galactosidase (E. co/ i(2) Glucose sub site

D-Glucose binds very poorly to free j-galac-tosidase but it does confer some binding forcesince lactose binds about 20-fold better thandoes o-galactose (Huber and Gaunt, 1983). Theglucose sub-site in the free enzyme has hydro-phobic character since galactosides with hydro-phobic aglycones bind very well (Yde and DeBruyne, 1978). However, minor structuralchanges in the hydrophobic aglycone moiety ofsubstrates and inhibitors result in altered bind-ing. For instance, ONPG, MNPG and PNPGshould bind with similar affinities because ofsimilar hydrophobicities, but the KS values (cal-culated from Tenu et al ., 1971) for ONPG andMNPG are about 10 times as large as forPNPG.

Glucose binds tightly after the glycosidicbond of lactose is cleaved (Huber et al., 1984)and the aglycone has diffused away. For thisbinding, a pyranose ring is required and thehydroxyl orientations at positions 2 and 4 areimportant. Also, the absence of a hydroxylgroup at position 6 results in a decrease inbinding at the glucose site.

REACTIONS OF /I-GALACTOSIDASE (2) Synthetic substrates(1) Lactose

Two reactions take place with lactose: hy-drolysis and intramolecular galactose transfer(Huber et al., 1976). The intramolecular transferreaction yields allolactose, the natural inducerof the lac operon. The reactions of the enzymeon lactose are summarized below (Fig. 1).

Figure 2 above is the accepted scheme ofthe P-galactosidase reaction with syntheticsubstrates in the presence of acceptors:

The constant k_, s for release of glucose whilek , is for binding glucose (many sugars andalcohols react instead of glucose and are accep-tors of galactose-that is why the a sub-scripts are used). The value of k , (identical for

It is assumed that the release of the aglyconeformed by the initial catalytic step is very fastand kinetically irrelevant. For ONPG, k , is2100 set- while for PNPG, k , is 120 set- . Thek , value is 1200 sect and, as stated earlier, it iscommon for all P-D-galactopyranosides. Theacceptor reaction is analogous to the reaction inthe lactose mechanism that forms allolactose.p-Nitrophenyl-cr-L-arabinopyranoside(PNPG without a 6 hydroxymethyl group)is a substrate (Marshall et al., 1977) and B-D-thiogalactopyranosides are very slowlyhydrolyzed (Wallenfels and Weil, 1972). Hy-drolysis of fluoro-p-D-galactopyranoside (vonHofsten, 1961) and of azidyl-B-o-galactopyra-noside (and other C-N bonds) also occurs(Sinnott, 1971; Sinnott and Withers, 1974).

ALLOLACTOSEFig. 1. The postulated reactions of B-galactosidase withlactose. The dots indicate that some sort of complex existswith the enzyme. E, B-galactosidase; GAL, galactose;

GLUC, glucose.

Fig. 2. The postulated reactions of /l-galactosidase onnitrophenyl substrates in the presence of an acceptor. Thedots indicate that some sort of complex exists with theenzyme. E, b-galactosidase; NPG, nitrophenyl substrate;GAL, galactose; NP, nitrophenyl product; A, acceptor;

GAL-A, galactosyl-acceptor adduct.

any /?-D-galactoside) is 1200 set- (Tenu et al .,1971). With lactose, k , is about 60 set- while k ,and k., are nearly identical (k, = 380 sect andk., = 360 set-) (Huber et al ., 1976, 1984). Thesimilarity of the values of these two constantscauses the initial ratio of glucose (or galac-tose) to allolactose production to be about one(Huber et al., 1975, 1976). Allolactose is, inreality, a transient product because it is also asubstrate of the enzyme (Huber et al., 1975).

(3) Reversion reactionsIf large amounts of D-galactose and D-

glucose are incubated with b-galactosidase,disaccharides form (Huber and Hurlburt, 1986).These reactions may be analogous to the for-mation of allolactose and, indeed, allolactose

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312 R. E. HUBER et alis the main product. Of a series of manydifferent compounds tried, only L-arabinose,r>-fucose and D-gala&al are able to replaceD-galactose and allow the reversion reactionto go. Molecules similar to D-galactose butwith alterations at the 2 position are notsubstrates for reversion despite binding aswell as D-galactose.

RE CTION MECH NISM

Figure 3 below is our model of the catalyticaction of the enzyme:This proposed mechanism involves non-co-valent interactions, general acid/base catalysisand electrostatic~covalent stabilization.(I) Non-covalent interactions

Non-covalent interactions of some type areimportant for catalysis. There is a definite corre-lation between the k,,, values of different sub-strates and the corresponding KS values. Forexample, the KS values for ONPG and MNPGare about 10 times as large as the KS for PNPGand the k,., values are also about 10 times higher

(Tenu et al., 1971). This is despite the fact thatpNP and oNP are good leaving groups andmNP is a poor leaving group. Also, the KSvalues for PNPG with ~-ga~actosidases withvarious substitutions for Glu-461 vary aboutproportionately with the kz values (Cuppleset al., 1990). /?-Galactosidase in some mannersacrifices its ability to bind substrates for cata-lytic energy.

It probably does this by stabilizing a tran-sition state that is planar at the anomeric end(that is why the model is drawn with a poor fitat the anomeric carbon of the substrate). Lac-tones have their C-l in plane with the ringoxygen and furanoses do not have a positionequivalent to C-l and both gaiactonolactoneand L-ribose (a furanose that resembles D-galac-tose) are very good inhibitors of fi-galactosidase(Huber and Brockbank, 1987). Changes at the6 position have the opposite effect. Binding ispoorer if either the C-6 hydroxyl or the wholehydroxy methyl group is absent (Marshall et al.,1977; Huber et al., 1983). A relevant findingregarding non-covalent interactions is thatG794D-/3-galactosidase has a k, value that is

ENZYMESUBSTRATE

16OH

ENZYMEGALACTOSE

Fig. 3. The positioning of Tyr is from Fritz et al. (1983) and Lehmann and Schlesselmann (1983).(Z)-3,7-Anhydro- I 2-deoxy-2-deuterio-D-galacto-act-2-eneitot was reacted with @-galactosidase and pro-tonation occurred from the top of the sugar (as normally viewed).

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/?-Galactosidase (E. coli) 313

several fold higher than the k , value of the simplest route for this is by proton addition.enzyme from wild-type E. col i (Martinez-Bilbao In addition, the hydration of D-galactalet al., 1991) and this mutated enzyme binds (Wentworth and Wolfenden, 1974; Lehmannplanar transition state analogs several fold bet- and Schroter, 1972) would most likely beter than does normal enzyme. On the other hand initiated by protonation. Finally, the modifi-it binds substrates and substrate analog inhibi- cation of Glu-461 with conduritol C cis-epoxidetors several times less well than does the normal (Herrchen and Legler, 1984) may require aenzyme. proton.

Sinnott and Souchard (1973) suggested thatconformational changes at the active site areinduced by some substrates and that this has aneffect on the activity. Presumably, these effectsare non-covalent in nature. The rates of reactionwith galactosyl pyridinium salts vary with thepKa of the leaving aglycones (Withers et al.,1978). This does not happen with other sub-strates. Withers et al. (1978) argued that confor-mation changes do not occur with thepyridinium salts (thus the dependency on thepKa values). With other substrates, the confor-mational changes might change the non-covalent interactions and obscure the effects ofthe pKa values.

Facilitation of reaction by acid catalysis wasestimated to be at least IO-fold when fl-D-galac-topyranosyl-azide was used to study the effect(Sinnott, 1971; Sinnott and Withers, 1974). Thisis a relatively small factor. Cleavage of galacto-syl pyridinium salt bonds (which cannot befacilitated by acid) is catalyzed slowly by B-galactosidase (Sinnott, 1973; Sinnott and With-ers, 1974; Withers et al., 1978). Also, asdiscussed above, catalysis does occur slowlywhen Tyr-503 is replaced.

We have recently studied the activity of /?-galactosidase with Mg2+ and with Mn2+ bound(unpublished). This was done with a series ofdifferent substrates and acceptors. Althoughdistinct differences in activity, binding, and ac-ceptor activity were found, these differencescould not be attributed to the differing chemicalproperties of these bivalent ions. For somesubstrates and acceptors, the activity wasgreater with Mg2+ while for others, the activitywas greater with Mn+. We concluded thatdifferences were due to differing conformationsthat caused different orientations at the activesite and, consequently, different non-covalenteffects.(2) General acid/base catalysis

Evidence that Tyr-503 may be an acid/basecatalyst has been discussed but there is otherevidence that acid catalysis occurs. p-Galactosi-dase is inactivated by /?-D-galactopyranosyl-methyl-p -nitrophenyl-triazene (Sinnott andSmith, 1976, 1978) and 2,6-anhydro-l-deoxy- l-diazo-D-glycero-L-manno-heptitol (Brockhausand Lehmann, 1976, 1978). Highly reactive cat-ion intermediates are probably involved. Thesecation intermediates should only form readily ifthere is proton addition (Isaacs and Rannala,1974). Also, Brockhaus and Lehmann (1977)synthesized 2,6-anhydro- 1 deoxy-D-galacto-hept-1-enitol and found that p-Galactosidaseconverted it to 1 deoxy-D-galactoheptulose. The

In the proposed mechanism, acid catalysisoccurs in the step with k , as the rate constantand base catalysis in the step with k,. WithpNPG (for which k , is rate limiting) and 3,5-dinitrophenyl-B-D-galactopyranoside (for whichk , is rate limiting) the activities of the alkalineside of the pH profiles decrease with inflectionpoints at 9.3 and 8.9, respectively (Sinnott andViratelle, 1973). The authors considered the twovalues to be the same. Studies done in a differentway by Huber et al . (1983) indicated, however,that k , and k , decrease with inflection points of9.4 and 8.6, respectively. The pH optimum ofthe V,,, is 7.6 for pNPG and lactose (Huberet al., 1976), (k, is rate determining for thesesubstrates) and the curve is very broad. The pHoptimum of the V,,, is 7.0 for oNPG (Huberet al., 1983) (k, is partially rate determiningfor this substrate) and for 3,5-dinitrophenyl-b-D-galactopyranoside (Sinnott and Viratelle,1973) (k, is rate determining) and the profilesare narrow. These findings suggest that k , andk , have different pKa values. There is a prob-lem, however. If a residue is an acid catalyst forthe breakage of the glycosidic bond (k,) and abase catalyst for the removal of a proton fromwater (k,), k, should decrease with pH and k ,should increase. But the values of k , and k , bothdecrease with pH. Another pH controlled fac-tor may be acting concurrently with base catal-ysis and may result in a large decrease of therate to mask a smaller base catalytic rateincrease. It is of interest in this regard that therate of reaction with E461D-P-galactosidase(which has very low activity at pH 7) increasesdramatically as the pH is raised (with an

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314 R E HUBER t al.inflection point of about 8.5) (Cupples et al.,1990). Possibly, for this modified enzyme, theother pH controlled effect that might be causingthe decrease in k, is absent or much decreasedand the base catalytic effect is unmasked.

Selwood and Sinnott (1990) have suggestedthat Mg*+ may be an el~trophilic catalyst. Theyfeel that the enzymes acid catalytic action is theprotonation of a Mg+-aglycone complex. Asstated previously, studies in our laboratorysuggest that Mg*+ is more likely to be importantonly in conformation.(3) E~ectrostati J~~ua~e~tcatalysis

Case et al. (1973) and Loeffler et al. (1974,1979) tested some positively charged galactoseanalogs which did not inhibit /I -galactosidase toa greater extent than did their unchargedcounterparts. On the other hand, Huber andGaunt (1982) found that amino sugars andalcohols are good inhibitors of fl-galactosidase(pa~ic~arly if their structures are similar tothat of D-galactose and if the amino group isattached to the anomeric carbon). Legler andHerrchen (1983) obtained similar results tothose of Huber and Gaunt and they stated thatthe inhibition probably arises from a donationof a proton to the amine so that an ion pairforms. Cupples et al. (1990) showed that re-moval of the negative charge at position 461 bysite specific substitution eliminates the bindingof the amino inhibitors. These results suggestthat the transition state involves (at the veryleast) a short-lived galactosyl cation stabilizedby Glu-461. Kinetic studies using substrateswith a deuterium at the ~-position of theanomeric carbin of galactose (Sinnott andSouchard, 1973) indicate that a galactosyl cat-ion develops and is stabilized by a negativecharge but those studies also showed that thisstabilized ion pair readily collapses to form acovalent bond to a nucleophile at the activesite. It was suggested that the covalentcomplex is in equilibrium with the ion pair andthat it is the cation component of this ion pairthat reacts with water. Rosenberg and Kirsch(1981) did studies with 0 and suggested thatdirect nucleophilic attack by a nucleophile onthe enzyme occurs with every substrate and thatthe formation of galactosyl cations only takesplace with substrates having good leavinggroups.~-Galactosi~ses with Asp, Gly, Gln, andLys substituted for Glu-461 are quite unreactivewith ONPG (Cupples et al., 1990). Substitution

by Asp causes both kl and k3 (ONPG) to besmall and the overall rate at pH 7.0 is very slow.Presumably, this is a result of the small size ofAsp. The k2 values (ONPG) of enzymes substi-tuted by Gly, Gln and Lys, are reasonably largebut the k, values are small (this, of course, alsoresults in a slow overall rate). When His wassubstituted, both k, and k, were relatively largefor ONPG and the reaction was relatively fast.None of these substituted enzymes (includingthe enzyme with His substituted), however, hadany significant activity when lactose was thesubstrate. Thus, the His substituted enzymemust have a small k, value with lactose (ofcourse the k, value does not change for differ-ent galactose substrates). We feel that thesefindings indicate that with ONPG, non-covalentinteractions and acid catalysis cause the for-mation of significant amounts of the galactosylcation intermediate even in the absence of anegative counterion because oNP is a goodleaving group. We think that the enzyme withHis at position 461 covalently traps the galacto-syl intermediate. For lactose, glucose is a poorleaving group and it is likely that non-covalentinteractions and acid catalysis are not able todrive the formation of the cation without ionpair and covalent stabilization and, since theinte~ediate is not formed in large amounts, itcannot be trapped by His. Generally, thesefindings support the idea that the formation ofa cation is induced by the combined effect ofnon-covalent interactions, acid catalysis andinduction of an ion pair by Glu-46 1. If directnucleophilic attack by Glu-461 were important,the reaction of the His substituted enzyme withlactose should have been fast since the 3N ofHis is about the same distance from its a-carbonas are the oxygens of the carboxyl group of Gluand an imidazole group is at least as good anucleophile as is a carboxyl group. This suggeststhat the role of Glu-461 is to induce the for-mation of a cation by electrostatic stabilization.Since there is now evidence that Glu-537 may beinvolved in nucleophilic attack (Gebler et al.,1992), it is possible that Glu-537 and Glu-461together induce the formation of a cation thatis then rapidly trapped by Glu-537. The water(or acceptor) then reacts with the covalent inter-mediate. That is how the mechanism is depictedon the above scheme. Glu-537 is probablylocated nearer to the anomeric carbon than inGlu-461 and thus forms the covalent bond. Thecation probably only has a fleeting existence.The fact that planar and charged analogs of

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B-Galactosidase (E. co/i) 315galactose are very good inhibitors and that yield of the disaccharides was improved by aGlu-461 is needed for inhibition by positively factor of two by constantly removing thecharged derivatives of galactose is strong product through a carbon-celite column con-evidence that galactosyl cations are at least nected to the reactor containing immobilizedshort-lived intermediates in the mechanism. E. coli /?-galactosidase (Hedbys, 1989).

BIOCHEMICAL AND BIOTECHNOLOGICALAPPLICATIONS REFERENCES(1) Production of low lactose milk and utilizationof whey lactose

Many adult persons cannot digest lactose.Production of low-lactose milk is carried outusing microbial /?-galactosidases (Gekas andLopez-Leiva, 1985). While E. co/i enzyme hasnot been used in commercial processes, con-siderable work with it has been done from theviewpoing of process development (Mosbachand Mattiasson, 1971; Inman and Hornby,1973; Khare and Gupta, 1987, 19881,b, 1990).Lactose hydrolysis is also of considerableimportance in utilization of whey becauseglucose and galactose have greater fermentationpotential (Kosaric and Asher, 1985).

Arrondo J. L. R., Muga A., Castresana J., Bemabeu C. andGoni F. M. (1989) An infrared spectroscopic study of/?-galactosidase structure in aqueous solutions. FEESLen. 252, 118-120.

Bader D. E., Ring M. and Huber R. E. (1988) Site-directedmutagenic replacement of glu-461 with gin in B-galactosi-dase (E. co/i): evidence that glu-461 is important foractivity. Eiochem. bi ophy s. Res. Commun. 153, 301-306.

Becker V. E. and Evans H. J. (1969) The influence ofmonovalent cations and hydrostatic pressure on /3-galac-tosidase activity. Biochim. biophys. Acta 191, 95-104.

Brockhaus M., Dettinger H.-M., Kurz G., Lehmann J. andWallenfels K. (1979) Participation of HO-2 in the cleav-age of p-galactosides by the p-galactosidase from E. coli.Curb. Res. 69, 264268.

Brockhaus M. and Lehmann J. (1976) 2,6-Anhydro-l-diazo-I-deoxy-D-glycero-L-manno-hepitol: a specific blockingagent for the active site of /I-galactosidase. FEBS Lerr. 62,154-156.

(2) Production of galactosyl adducts using thetransgalactosylation reactionj?-Galactosidase causes the formation of tri-and tetra-saccharides starting with lactose (Hu-ber et al., 1976). It has been found that thenature of the products is quite dependent on thesource of the enzyme (Prenosil et al., 1987). Itwas found that b-galactosidase from E. coliyielded only one isomer (j?-D-galactopyranosyl-(1-6)-N-acetyl-galactosamine) when incubatedwith lactose and N-acetyl-galactosamine (Hed-bys et al., 1984). Recently Huber and Chivers(1994) showed that j?-D-galactosyl adducts formwhen oNPG is incubated with various nucle-ophiles and with P-galactosidases with site-specific substitutions for Glu-461. This also haspotential for the synthesis of a-r>-galactosyladducts. If an adduct with good leaving abilitywere to be added in the p-orientation by theaction of this site-specific mutant and if this wasthen reacted with a strong nucleophile todisplace it, an cr-adduct should form.

Brockhaus M. and Lehmann J. (1977) The conversion of2,6-anhydro-I-deoxy-D-galacfo-hept-I-enitol into I-de-OXy-D-ga/aCfO-heptulose by fi-~galactosidase. Carb. Res.53, 21-31.

Brockhaus M. and Lehmann J. (1978) Ester and sulfoniumsalt formation in the active-site labeling of fi-D-galactosi-dase from Escherichia coli by 2,6-anhydro-l-deoxy-l-di-azo-D-glycero-L-manno-heptitol. Carb. Res. 63, 301-306.

Burchhardt G. and Bahl H. (1991) Cloning and analysis ofthe B-galactosidase-encoding gene from Closfridium fher-mosulfurogenes EM 1. Gene 106, 13-19.

Buvinger W. E. and Riley M. (1985) Nucleotide sequence ofKl ebsiell a pneumoniae l ac genes. J. Bacferiol. 163,850-857.

(3) Synthesis of disaccharides by reversal ofhydrolysis

Case G. C., Sinnott M. L. and Tenu J. P. (1973) The rol eof magnesium ions n p-galactosidase-catalysed hydrolysis:studies on charge and shape of the /I-galactopyranosylbinding site. Eiochem. J. 133, 99-104.

Cohn M. (1957) Contributions of studies on the P-galactosi-dase of Escherichia co/i to our understanding of enzymesynthesis. Bacferiol. Rev. 21, 140-168.

Cohn M. and Monod J. (1951) Purification and propertiesof the /?-galactosidase (lactase) of Escherichia coli.Biochim. biophys. Acfa 7, 153-174.

Cupples C. G. and Miller J. H. (1988) Effects of amino acidsubstitutions at the active site in Escherichia coli p-galac-tosidase. Genetics 120, 637-644.

Cupples C. G., Miller J. H. and Huber R. E. (1990)Determination of the roles of Glu-461 in fi-galactosidase(Escherichia co/i) using site-specific mutagenesis. J. biol.Chem. 265, 55 12-55 18.As already discussed, the formation of galac-topyranosides from the component monosac-

charides is catalyzed by /?-galactosidases byusing high sugar concentrations to shift theequilibrium (Huber and Hurlburt, 1986). The

David S., Stevens H., van Riel M., Simons G. and de VosW. M. (1992) Leuconosroc lacifs fl-galactosidase is en-coded by two overlapping genes. J. Bacferiol. 174,4475448 I.

Edwards L. A. and Huber R. E. (1986) A detailed examin-ation of the iodination of /I-galactosidase: stoichiometric

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316 R. E. HUBER et alinactivation by nonspecific iodination. Biochem. Cell immobilized P-galactosidase. Biochem. biophys. Res.Biol. 64, 523-527. Commun. 123, 8-15.

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