CHAPTER 10: REGULATORY STRATEGIESocw.sogang.ac.kr/rfile/2014/Biochemistry I/Chapter10... · 2014....

CHAPTER 10: REGULATORY STRATEGIES

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INTRODUCTION

The activity of enzymes must often be regulated so that they

function at the proper time and place.

Enzymatic activity is regulated in five principal ways:

1. Allosteric Control

• Enzyme activity is controlled by the binding of small signal

molecules at regulatory sites

• Allosteric proteins show the property of cooperativity: activity

at one functional site affects the activity at others

• Aspartate transcarbamoylase (ATCase)

• Hemoglobin

CHAPTER 10

INTRODUCTION

2. Multiple Forms of Enzymes

• Isozymes are homologous enzymes within a single organism

- Catalyze the same reaction

- Slightly different in structure and catalytic/regulatory

properties

- Expressed in a distinct place or at a distinct stage of

development

CHAPTER 10

3. Reversible Covalent Modification

• Alters the catalytic properties of many enzymes

- Phosphorylation by protein kinases

- Dephosphorylation by protein phosphatases

- Protein kinase A

INTRODUCTION

4. Proteolytic Activation

• Activation of proenzymes (zymogens) by proteolytic

cleavage

- Chymotrypsin, trypsin, and pepsin are activated by this

mechanism

- Blood clotting is due to a cascade of zymogen activations

CHAPTER 10

5. Controlling the Amount of Enzyme Present

• Enzyme activity is regulated by adjusting the amount of

enzyme present

- This regulation usually takes place at the level of

transcription

10.1 ASPARTATE TRANSCARBAMOYLASE

The enzyme catalyzes the 1st step in the biosynthesis of

pyrimidines:

• The condensation of aspartate and carbamoyl phosphate

• The committed step in the pathway for pyrimidine nucleotide

such as CTP

CHAPTER 10

Fig 10.1 ATCase reaction.


It was found that ATCase is inhibited by CTP

Feedback inhibition

• The inhibition of an enzyme by the end product of the

pathway

CHAPTER 10

Fig 10.1 CTP inhibits ATCase.

Allosteric regulation

• CTP is structurally quite

different from the substrates

• CTP must bind to a site

distinct from the active site

SIGMOIDAL KINETICS

The dependence of the reaction rate on [Asp]

• Sigmoidal curve

• Cooperativity – the binding of substrate to one active site

increases the activity at the other active sites


Fig 10.3 ATCase displays

sigmoidal kinetics.

• Does not follow Michealis-Menten

kinetics

The majority of allosteric enzymes

display sigmoidal kinetics

• Cooperation between subunits in

hemoglobin

CATALYTIC AND REGULATORY SUBUNITS

ATCase can be separated into regulatory (r) and catalytic (c)

subunits by treatment with p-hydroxymercuribenzoate (p-HMB)

• Catalytic subunit (c3), three chains (34 kd each)

• Regulatory subunit (r2), two chains (17 kd each)


Fig 10.4 Modification of cysteine residues. Fig 10.5 Ultracentrifugation studies of ATCase.

Native p-HMB treated

CATALYTIC AND REGULATORY SUBUNITS

The larger subunit displays catalytic acitivity; unresponsive to

CTP, not sigmoidal kinetics – catalytic subunit

The smaller subunit can bind CTP; no catalytic acitivty –

regulatory subunit

The subunits combine rapidly when they are mixed

2 c3 + 3 r2 → c6r6

The reconstituted enzyme has the same structure and

allosteric/catalytic properties as those of the native enzyme


STRUCTURE OF ATCASE

Two catalytic trimers are stacked one on top of the other

Significant contacts between the catalytic and the regulatory

subunits


4-Cys bound

Fig 10.6 Structure of ATCase.

STRUCTURE OF ATCASE

A bisubstrate analog, N-(phosphonacetyl)-L-aspartate (PALA)

was used for the ATCase-substrate analog complex structure

• PALA is a potent inhibitor for ATCase


Fig 10.7 PALA, a bisubstrate analog.

STRUCTURE OF ATCASE

The structure of the ATCase-PALA complex

• PALA binds at site lying at the boundaries between pairs of c

chains within a catalytic trimer


Fig 10.8 The active site of ATCase.

STRUCTURE OF ATCASE

Remarkable change in quaternary structure on PALA binding

ATCase shows two distinct quaternary forms: the T (tense)

state and the R (relaxed) state


Fig 10.9 The T-to-R state transition

in ATCase.

STRUCTURE OF ATCASE

How can we explain the enzyme’s sigmoidal kinetics?

In the absence of substrate, almost all the enzyme molecules

are in the T state

• Low affinity for substrate; low catalytic activity

The substrate binding to one active site

• Increases the likelihood that the entire enzyme shifts to the R

state with higher affinity

• Conversion of the enzyme into the R state causes more

substrate to bind to the active site

• Cooperativity


STRUCTURE OF ATCASE

The sigmoidal curve can be pictured as a composite of two

Michealis-Menten curves

The T-to-R transition is taking place within a narrow range of

substrate concentration


Fig 10.8 Basis for the sigmoidal curve.

• Makes it possible to respond

to small changes in substrate

concentration

CTP EFFECTS ON THE T-TO-R EQUILIBRIUM

CTP inhibits the action of ATCase

Structure of CTP-bound ATCase

• The enzyme is in the T state

• CTP binds to the regulatory domain

• The binding site is more than 50 Å from

the active site


Fig 10.11 CTP stabilizes the T state.


The binding of CTP shifts the equilibrium toward the T state

• Stabilizes the T state

• Decreases net enzyme activity

• Increases the initial phase of the sigmoidal curve


Fig 10.13 Effect of CTP on ATCase kinetics.

Fig 10.12 The R state and the T state are in equilibrium.


ATP increases the reaction rate at a given Asp concentration

• Competes with CTP for binding to regulatory sites

• High ATP means high purine: needs to make more pyrimidine

for balance


Fig 10.14 Effect of ATP on ATCase kinetics.

• High ATP means high energy

for mRNA synthesis and DNA

replication: leads to the

synthesis of more pyrimidines

http://www.youtube.com/watch?v=5aW0C3-IHVo

10.2 REGULATORY STRATEGIES IN ISOZYMES

Isozymes are enzymes that differ in AA sequence yet catalyze

the same reaction

Have different kinetic parameters or respond to different

regulatory molecules

Permits the fine-tuning of metabolism to meet the needs of a

given tissue or developmental stage

Lactate dehydrogenase

• Involved in glucose synthesis and metabolism

• Two isozymic peptides exist: H isozyme and M isozyme;

75% identical sequence

• Functions as a tetramer: many different combinations exist

CHAPTER 10

10.2 REGULATORY STRATEGIES IN ISOZYMES

The isozymes (H4 & M4) are functionally different

• H4: inhibited by high levels of pyruvate; functions in aerobic

environment

• M4: no inhibition by high levels of pyruvate; functions in

anaerobic environment

• H2M2 has intermediate properties

CHAPTER 10

Fig 10.16 Isozymes of lactate dehydrogenase.

The rat heart LDH isozyme profile

Days before (-) & after (+) birth

M4

H4

The tissue-specific forms of lactate dehydrogenase

in adult rat tissues

10.3 COVALENT MODIFICATION

CHAPTER 10

KINASES AND PHOSPHATASES

Phosphorylation is a regulatory mechanism used in every

metabolic process in eukaryotic cells

• 30% of eukaryotic proteins are phosphorylated

• Catalyzed by protein kinases

• Protein kinases are one of the largest protein families: more

than 500 protein kinases in human beings

• Fine-tuned regulation according to a specific tissue, time, or

substrate

• ATP is the most common donor

• Ser, Thr, and Tyr are the acceptors





Tyrosine kinases

• Play pivotal roles in growth regulation

• Mutations are observed in cancer cells

Ser/Thr kinases



Substrate specificity of Ser/Thr kinases

• Some kinases phosphorylate a single protein or several

closely related ones

• Multifuncational kinases modify many different targets

- Recognize the consensus sequence, Arg-Arg-X-Ser/Thr-Z;

X a small residue; Z, a large hydrophobic residue



Protein phosphatases remove

phosphoryl groups attached to proteins

• Turn off the signaling pathways

activated by kinases

• One class of conserved phosphatases

(PP2A) suppresses the cancer-

promoting activity of certain kinases


Irreversible / unidirectional

Take place only by enzymes

PHOSPHORYLATION IS HIGHLY EFFECTIVE

Phosphorylation is a highly effective means of regulating the

activities of target proteins for several reasons:

1. The free energy of phosphorylation is large

• 20 ~ 30 kJ/mol (about 5 kcal/mol)

• A free energy change of 5.69 kJ/mol (1.36 kcal/mol)

corresponds to a factor of 10 in an equilibrium constant

• Phosphorylation shifts the equilibrium by 104

2. Adds two negative charges to a modified protein

• These charge can cause a large conformational change

• Such structural changes can markedly alter substrate binding

and catalytic activity





3. A phosphoryl group can form three or more H-bonds

• Can make specific interaction

4. Phosphorylation and dephosphorylation can take place in less

than a second or over a span of hours

• The kinetics can be adjusted to meet the timing needs of a

physiological process





5. The effects of phosphorylation can be highly amplified

• A single activated kinase can phosphorylate hundreds of

target proteins in a short interval

6. ATP is a cellular energy currency

• The use of ATP as a phosphoryl-group donor links the energy

status of the cell to the regulation of metabolism


CYCLIC AMP ACTIVATES PROTEIN KINASE A

Cyclic AMP (cAMP) is an

intracellular messenger formed by

the cyclization of ATP

cAMP (>10 nM) activates a key

enzyme, protein kinase A (PKA)

The activated PKA alters the

activities of target proteins by

phosphorylation

Most effects of cAMP in eukaryotic

cells are achieved through the

activation of PKA


PKA consists of two kinds of subunits:

• A 49-kD regulatory (R) subunit and a 38-kD catalytic (C) subunit

• In the absence of cAMP, PKA exists in a R2C2 form

The binding of cAMP to the R subunit relieve its inhibition of the C subunit

• Each R chain contains the sequence Arg-Arg-Gly-Ala-Ile

(pseudosubstrate sequence), which occupies the catalytic site of C

• The binding of cAMP allosterically removes the sequence resulting in

the activation of C

CYCLIC AMP ACTIVATES PROTEIN KINASE A10.3 COVALENT MODIFICATION

Fig 10.17 Regulation of PKA.

THE CRYSTAL STRUCTURE OF PKA

The X-ray crystal structure of PKA complexed with ATP and a

20-residue peptide inhibitor


Two lobes: the smaller lobe,

contacts with ATP-Mg2+; the

larger lobe, binds to the peptide

substrate

Residues 40 to 280 are

conserved in all known protein

kinases

Fig 10.18 PKA bound to an inhibitor.

THE CRYSTAL STRUCTURE OF PKA

The bound peptide in the structure occupies the active site


Two guanidinium groups

interact with three carboxylates

in the active site

Two Leu make a hydrophobic

site to accommodate the Ile of

the peptide

Fig 10.19 Binding of pseudosubstrate to protein kinase A.

The binding of cAMP allosterically

blocks this interaction resulting in

the activation of C

GLEEVEC

A tyrosine-kinase inhibitor used in the

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Received FDA approval in May 2001

Imatinib was one of the first cancer

therapies; a paradigm for research in

cancer therapeutics

Imatinib has been cited as the first of

the exceptionally expensive cancer

drugs


Time magazine cover of 28

May 2001 detailing Glivec as a

'cure' for cancer.

//upload.wikimedia.org/wikipedia/en/5/59/Time_cover_on_Glivec_28_May_2001.png

//upload.wikimedia.org/wikipedia/commons/9/9b/Imatinib2DACS.svg

10.4 REGULATION BY PROTEOLYTIC CLEAVAGE

Specific proteolysis is a common means of activating

enzymes

• The inactive precursor is called a zymogen or a proenzyme

Examples of regulation by proteolytic cleavage

1. Digestive enzymes

CHAPTER 10



2. Blood clotting – thrombin, cascade

3. Some protein hormones

• Insulin is derived from proinsulin by proteolytic cleavage

4. Developmental processes: conversion of procollagenase into

collagenase

• The metamorphosis of a tadpole into a frog: resorption of

large amounts of collagen from the tail

• Break down of collagen in a mammalian uterus after delivery

• The conversion is precisely timed in these remodeling

processes

CHAPTER 10



5. Apoptosis (programmed cell death)

• Eliminates damaged or infected cells and controls the

shapes of body parts in the course of development

• Apoptosis is mediated by caspases, proteolytic enzymes

• Caspases are generated from procaspases by proteolytic

cleavage

• Caspases function to cause cell death in most organisms

ranging from C. elegans to human beings

CHAPTER 10

CLEAVAGE OF CHYMOTRYPSINOGEN

Chymotrypsinogen

• Consisting of 245 AAs

• Synthesized in the

pancreas

• Inactive form of

chymotrypsin

• Cleaved by trypsin and

converted into two peptides

• Self-cleaved to produce

three peptide chains and

two dipeptides


Fig 10.21 Proteolytic activation of chymotrypsin.

HOW THE CLEAVAGE ACTIVATES THE ZYMOGEN? The cleavage generates a new interaction between the N-terminal

of Ile16 and the side chain of Asp194


The new interaction triggers a

number of conformational

changes

• Residues 187, 192, and 193

• The changes generates the

substrate-specificity site for

hydrophobic groups

• Generation of the oxyanion

hole

• Highly localized

conformational change Fig 10.22 Conformations of chymotrypsinogen

(red) and chymotrypsin (blue).

TRYPSIN IS THE COMMON ACTIVATOR

Trypsin is the common activator of all the pancreatic zymogens

• Trypsinogen, chymotrypsinogen, proelastase,

procarboxypeptidase, and prolipase

• The formation of trypsin by enteropeptidase is the master

activation step.


Fig 10.23 Zymogen activation by

proteolytic cleavage.

TRYPSIN IS THE COMMON ACTIVATOR

The activity of trypsin is controlled by a pancreatic trypsin inhibitor

• The inhibitor is a 6-kd protein


Fig 10.24 Interaction of trypsin with

its inhibitor.

The dissociation constant of the complex is 0.1 pM

• Substrate analog

• Preorganized structure

• Cleavage rate of the inhibitor by

trypsin is very slow (several

months of half-life)

BLOOD CLOTTING

Enzymatic cascades in biochemical systems

• Achieve a rapid response; an initial signal triggers a series of

steps each of which is catalyzed by an enzyme

• Activation of 10 enzymes by an enzyme can activate 104

enzymes in 4 steps

Blood clots are formed by a cascade of zymogen activations

• Small amounts of the initial factors suffice to trigger the cascade


BLOOD CLOTTING


Fig 10.26 Blood clotting cascade.

Blood clotting is achieved by the

interplay of the intrinsic, extrinsic,

and final common pathways

Begins with the activation of factor

XII by contact with abnormal

surfaces produced by injury

Triggered by trauma, which releases

tissue factor (TF)

TF forms a complex with VII, which

initiates a cascade-activating

thrombin

Inactive form, in red

Active form, in yellow

Activated by thrombin, with *

FLUORESCENT PROTEINS

History

Structure

Mechanism of the fluorescence

Mechanism of the various colors

Applications

Due: the day of the 1st exam (will be early April)

HOMEWORK #1

http://4.bp.blogspot.com/_iirq14WQO4w/SOzM6SoYPAI/AAAAAAAAAQ8/yXvrHBiH2zo/s1600-h/GFP_aequorea_victoria-1.jpeg

http://upload.wikimedia.org/wikipedia/commons/e/e4/GFP_structure.png

FLUORESCENT PROTEINS

HOMEWORK #1

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