Chapter 7: Enzyme Regulatory Strategies

Enzymatic activity is regulated in 5 principal ways

1. Allosteric control by regulatory molecules
2. Alternate enzymes that catalyze the same reactions but
exhibit different catalytic and regulatory properties
3. Reversible covalent modifications
4. Irreversible proteolytic cleavage to yield an active
enzyme product
5. Controlling the amount of enzyme present

Allosteric regulation enables

Control of metabolic pathways

Pyrimidines and purines

Aromatic groups that compose the unique bases found in DNA and RNA

Aspartate transcarbamoylase (ATCase)

Catalyzes the first step in pyrimidine biosynthesis

Committed step

Step in the pathway where the products of the reaction are committed to the ultimate synthesis of end products in the pathways

Committed step is irreversible under

Cellular conditions

Committed step is catalyzed by

Allosteric enzymes

What catalyzes the committed step in pyrimidine synthesis

ATC

What inhibits ATCase

CTP

What is feedback inhibition?

inhibition of an enzyme by the end product of the pathway

What is an example of feedback inhibition

ATCase inhibition by CTP

CTP inhibits ATCase ensures pathway intermedates

Are not needlessly formed

CTP is an

Allosteric inhibitor

CTP is structurally

Different from the reaction substrates

CTP is structurally different from

The reaction substrates

Allosteric (or regulatory) sites

Sites distinct from the active site at which substrate binds

CTP is an allosteric inhibitor that

exerts its effects by binding at a distinct allosteric site on ATCase

Many allosterically regulated enzyme do or do not follow Michaelis-Menten kinetics?

Do not follow

ATCase, like the majority of allosteric enzymes

Display sigmoidal kinetics

Sigmoidal curves result from

Cooperation between subunits

ATCase consists of

Separable catalytic and regulator subunits

Native ATCase consits of

6 catalytic © and six regulatory ® subunits (c6r6) organized as 2 catalytic trimers and 3 regulator dimers

Isolated subunits combine

Rapidly when mixed to from c6r6

p-Hydroxymercuribenzoate Reacts

with
Crucial Cysteine Residues in ATC

Treatment of ATCase with
p-hydroxymercuribenzoate
dissociates the enzyme
into

• a catalytic subunit
  consisting of three chains
  (c3).

• - a regulatory (r) subunit of
  two chains (r2).

Allosteric Interactions in ATCase Are
Mediated by

Large Changes in Quaternary Structure

Two catalytic timers are stacked upon one another, linked by 

The three regulatory dimers

Significant contacts exist between

The catalytic and regulatory subunits

Significant contacts exost between the catalytic and regulatory subunits

A zinc ion is critical for the interactions of the r chain with the c chain

What is an analog of an intermediate along the ATCase catalytic pathway

PALA

PALA is

A potent competitive inhibitor of ATCase

PALA binds to

And blocks the active sites

Where does PALA bind to

Binds at the boundaries between the pairs of c chains within a catalytic trimer

Each catalytic timer contributes

3 active sites to the enzyme

ATCase exists in

2 conformations

Binding of PALA causes

Structural changes that convert the compact, less active tense (T) state into the expanded, more active relaxed (r ) state

ATCase exists in an equalibrium between

The T state and the R state

What is allosteric coefficient (L)

Ratio of the concentration of enzyme in the T state to that in the R state

Describe the T state

• Has low affinity for substrate

• Has low catalytic activity

• Is favored in the absence of substrate

Describe the R state

• Has higher affinity for substrate

• Is the most active form

• Is favored upon binding of substrate

What is cooperativity

Property by which the subunits cooperate with one another

What is homotropic effects?

Cooperative effects of substrates on allosteric enzymes

What does homotropic effects act on?

Enzyme active sites

What does homotropic effect generate?

The sigmoidal curve

What does homotropic effects impact?

ATCase activity by altering the T/R ratio

What are limiting models for cooperativity?

• ATCase adhered to the concerted (‘all or none') mechanism for allosteric enzymes

• Most other allosteric enzymes exhibit features of both the concerted and the sequential model

ATCase adheres to

The concerted mechanism for allosteric enzymes

• The entire enzyme is converted from T into R

Most other allosteric enzymes exhibit

Features of both the concerted and the sequential model

The sigmoidal curve for ATCase is

A composite of two Michaleis-Menten cruves

What are the two michaleis-menten cruves that the sigmodial curve of ATCase is made of?

• The less active T state at low [S]

• The more-active R state at high [S]

Allosteric enzymes display

Threshold effects

The transition of the threshold effects of allosteric enzymes occur

Within a narrow range of substrate concentration

What is the threshold effect?

The activity of allosteric enzymes (red line0 is more sensitive to changes in [S] near Km than are Michaelis-Menten enzymes (blue line) with the same V max

Allosteric regulators modulate

The T-to-R equalibrium

ATCase is in the what state when bound to CTP?

T state

The CTP binding site exists in

Each regulatory chain in a domain that does not interact with the catalytic subunit

What stabilizes the T state?

CTP

The R state and the T state are in

Equalibrium

Describe the binding of CTP to the regulatory site of ATCase

• Favors the T state

• Decreases net enzyme activity

• increases L from 200 to 1250

CTP and ATP impact ATCase kinetics..

Differently

What is an allosteric activator of ATCase

ATP

Describe ATP being an allosteric activator of ATCase

• Binds to the same site as CTP

• Favors the R state

• Increases net enzyme activity

What are heterotropic effects?

Effects of nonsubstrate molecules on allosteric enzyems

Heterotropic effects bind

At sites other than the active site

Heterotropic effects shift

The Km

Heterotropic effects impact

ATCase activity by altering the T/R ratio

What provides a means of regulation specific to dinsint tissues and developmental stages?

Isoenzymes

What are isoenzymes?

Enzymes that differ in amino acid sequence that catalyze the same reaction

Isoenzymes typically display

Different kinetic parameters or respond to different regulatory molecules

Isoenzymes are encoded by

Different genes

Isoenzymes may be expressed 

In a tissue-specific or development state specific pattern 

What dehydrogenases isoenzymes?

Lactase

What is Lactate Dehydydrogenase (LDH)?

Tetrameric protein that catalyzes a step in anaerobic glucose metabolism and glucose synthesis

What are the 2 isozymic polypeptide chains for LDH that humans have?

• H protein is highly expressed in heart muscle

• M protein is highly expressed in skeletal muscle 

How many isoenzymes are there of lactate dehydrogenase?

5

Isoenzyme proportions change

Throughout rat hear development as tissue switches from an anaerobic to aerobic environment

LDH isoenzyme content varies by 

Tissue type 

Covalent modification is

A means of regulating enzyme activity

What is covalent modificatIon?

Covalent attachement of a modifying functional group to an enzyme

Most covalent modifications are

Reversible

What are exmaples of covalent modifications?

Phosporylation, dephosporylation, acetylation, deacelatyion

What contnrol the extent of protein phosphorylation?

Kinases and phosphatases

What are protein kinases?

Catalyze the phosphorylation of protein substrates by attaching a phosphoryl group to a Ser, Thr, or Tyr residue

What is the most common phosporyl group donor?

ATP

What are regulated by reversible phosphorylation?

Only intracellular proteins

Individual kinases may recognize

Many related amino acid sequences or consequences

Are the exact sequences of individuals kinases being recognized required?

No, X and Z can vary

protein kinase A recognizes Arg-Arg-X-Ser-Z or
Arg-Arg-X-Thr-Z.. What is X?

Small residue

protein kinase A recognizes Arg-Arg-X-Ser-Z or
Arg-Arg-X-Thr-Z.. What is Z?

Large hydrophobic resideu

protein kinase A recognizes Arg-Arg-X-Ser-Z or
Arg-Arg-X-Thr-Z.. What is Ser or Thr?

The site of phosphorylation

What are protein phosphatases?

Catalyze the removal of phosphoryl group attached to proteins by hydrolyzing the bond attaching the phosphoryl group

The reactions catalyzed by kinases and phosphatases are?

Irreversible. under cellular conditions

Are phosphorylation and dephosphorylation reactions the reverse of one another?

NO

Reactions take place how in the absense of enzymes?

At a negllible rate

Target proteins cycle between

Unphosphorylated and phosphorylated forms

What is a means of regulating the activities of target proteins?

Phosphorylation

Why is phosphorylation highly effective?

• the free energy of phosphorylation is large.
  – a phosphoryl group adds two negative charges to a protein,
  potentially altering electrostatic interactions.
  – a phosphoryl group can form 3+ hydrogen bonds.
  – kinetics of phosphorylation and dephosphorylation can be
  adjusted to meet timing needs of a particular process.
  – phosphorylation can be used to amplify signals.
  – ATP is the cellular energy currency.

How does cyclic AMP activate protein Kinase A

By altering the quaternary structrue

What induces the figh or flige responses in muscles?

Epinephrine (adrenaline)

Epinephrine triggers the

Formation of the intracellular messenger cyclic AMP (cAMP)