Lecture 11. Biochemistry 3521 - Fordham University - 1999

---

Some of this material is taken and modified from:

Biochemistry 3033 at Florida International University (kind permission of Dr. Kelsey Downum)

(Note that the Enzyme Kinetic Figures were kindly provided by Kelsey Downum)

Biochemistry 242 at Illinois State University (kind permission of Dr. Reef Morse)

Biochemistry 100B at UCSC (Dr. Robert Fink)

---

Version: 2/15/99

---

TOPICS TO BE COVERED

A. Enzyme Inhibition

B. Enzyme regulation and allosterics 

---

Lecture 12

A. Insights into enzyme catalysis and mechanisms

B. Examples of mechanisms of enzyme regulation

---

---

A. ENZYME INHIBITION

---

General Comments

In general enzyme inhibition involves the inhibitor interacting with the enzyme (covalently or non-covalently) so as to reduce or abolish its catalytic activity. Key components of metabolic pathways and their regulatory systems are natural inhibitors. Thus, the regulation of metabolism can be viewed in part as mechanisms of inhibition.

Basis of most chemotherapeutic regimes is enzyme inhibition. Many health disorders can be controlled, in principle, by inhibiting selected enzymes. Two examples include methotrexate and FdUMP, common anticancer drugs which inhibit enzymes involved in the synthesis of thymidine and hence DNA, and penicillin, an antibiotic which inhibits the enzymes involved in the synthesis of the bacterial cell-wall.

Other important enzyme inhibitions are those caused by nerve gases and by heavy metal poisoning.

The study of enzyme inhibition is important for many reasons, among them:

• useful in elucidating kinetic mechanisms
• useful in elucidating the nature of enzyme-substrate intermediates and complexes and the chemical mechanism of catalytic action
• as drugs for target enzymes = Rational Drug Design
• providing information regarding metabolic regulation and control of metabolic enzymes.

General Types:

Reversible

Irreversible

"Suicide"

• Reversible
• competitive
• non-competitive
• uncompetitive
• mixed

• Irreversible
• covalent modification or denaturation of the enzyme
• An example is a thiol containg enzyme reacting with ICH2COOH (iodoacetate)(see Fig. 8-18)
• Enz-SH + ICH2COOH Enz-S-CH2COOH + I-
• This results in covalent modification of SH and is irreversible
• Affinity labels or active-site directed modifying agents
• Their structure directs them to the active site and they typically modify amino acid R-groups at the site
• An example we will discuss next time (if time permits) is TLCK and trypsin.

 "Suicide" substrates or mechanism-based inhibitors

The enzyme converts an innocuous substrate into an inactivator during the catalytic process

• e.g. penicillin sulfone reacting with beta-lactamase (see supplementary information provided by Dr. Fink at UCSC on penicillins).

• Competitive inhibition
• Raises Km only (intercept in L-B plot)
• S and I compete for same binding site
• Non-competitive inhibition
• Lowers Vmax only (slope in L-B plot)
• I binds at a site distinct from that at which the S binds
• Uncompetitive inhibition
• Both V_max & K_m decrease
• I binds to ES complex, but not free E
• Mixed inhibition
• Affects both Km and Vmax
• Formation of an ESI complex which does not break down to products at a significant rate

• Competitive Inhibitors - most common type
• It is usually based on the fact that the inhibitor structure resembles that of the substrate and the inhibitor competes with the substrate for the substrate binding site. Inhibitor physically blocks the binding of the substrate. Competitive inhibition can also occur in allosteric enzymes where the inhibitor binds to a distant site, and causes a conformational change which affects the structure of the active site and prevents substrate binding from occurring (called or considered nonclassical competitive inhibition by some people).
• One interesting aspect is that this type of inhibition (competitive) can be reversed if a sufficiently high concentration of substrate is added.
• I competes w/S for the active site of the enzyme (Fig. 8-19; 8-20)

• V_max stays the same, but K_m increases (M & M plot; L-B plot)

E + S <--> ES <--> E + P 
+ 
I <--> EI

• Once bound, I prevents S from being converted into P
• Inhibition can be reversed by increasing [S]
• Competitive inhibitors generally resemble the 3-D structure of the substrate
• Competive inhibition can be analyzed by L-B (see Fig. 8-20)
• Supplementary information
• v = VmaxS/{Km(1 + I/Ki) + S}
• Thus, in the presence of the inhibitor the observed Km actually equals Km(1 + I/Ki).
• By measuring the apparent Km as a function of inhibitor concentration one can determine Ki.
• Ki is a measure of the affinity of the enzyme for the inhibitor.
• Ex. Succinate Dehydrogenase substrate = succinate
• malonate & OAA structurally resemble succinate (they are also intermediates of the TCA cycle)
• neither is oxidized by the enzyme (electrostatic attraction)

• Non-Competitive Inhibitors - typically observed in allosteric enzymes (see Fig. 8-21)
• Inhibitor binds to a site other than the active site (often allosteric site - located somewhere else on the enzyme)
• Addition of excess substrate will not overcome the inhibition
• Physically, binding of the inhibitor leads to a change in the enzyme's catalytic properties, making it less effective
• The alternative which produces the opposite effect is an activator (positive) -- this might increase conversion of substrate to product or decrease K_m
• K_m stays the same, but V_max decreases (M & M plot; L-B plot)

           I <--> ESI 
           + 
E + S <--> ES <--> E + P 
+ 
I <--> EI

v = VmaxS/(1 + I/Ki)/{Km + S}

• Alter an enzyme so that reversible inactivation of the catalytic site results
• Inhibitors bind to both E & ES to form EI & ESI complexes
• Generally, are often metabolic intermediates that bind & regulate enzymes in metabolic pathways (i.e., feedback inhibition/allosterics)

• Uncompetitive Inhibitors
• I binds to ES complex, but not free E
• V_max affected since conversion of ES to E + P inhibited
• K_m also decreases because the equilibrium for formation of ES and ESI are shifted toward the complexes by binding of I
• Typically only seen in multisubstrate reactions
• Therefore = Both V_max & K_m decrease with ratio of V_max /K_munchanged (L-B plot)

E + S <--> ES <--> E + P
                 +
                  I <--> ESI
• Mixed Inhibitors -- also fairly common (variant of un and non)
• Formation of an ESI complex which does not break down to products at a significant rate
• Both K_m and V_max are affected to different degrees
• v = VmaxS/(1 + I/Ki)/{Km(1 + I/Ki') + S}
• Ki = (ES x I)/ESI and Ki' = (E x I)/EI

• Some examples
• Diisopropylfluorophosphate (DFP ) - irreversibly binds to catalytic sites that contain Serine (OH group) (for example, acetylcholinesterase, trypsin, chymotrypsin, etc.) -- serine proteases or esterases
• Nerve gas (DFP) irreversibly inhibits serine esterase acetylcholinesterase which catalyzes hydrolysis of acetylcholine, a neurotransmitter
• Inhibition causes paralysis due to lack of inactivation of neurotransmitter follow nerve cell binding = constant nerve firing
• Malathion - specific for insect acetylcholinesterase (organophosphorus inhibitor)
• Cyanide - binds to transition metals that are used as cofactors (Fe, Cu, Zn, etc.)
• Iodoacetate - reacts with cyteine SH (C), imidizol (H), carboxy & R-S-R groups (M)

•  Irreversible inhibitors are useful in probing active sites of enzymes

• Covalent modification of an enzyme may lead to loss of activity if
• An essential catalytic group is modified i.e. blocked
• Substrate binding is sterically hindered
• The modification leads to some conformational distortion of the enzyme or mobility restraint.
• Many reactive chemicals will modify reactive amino acid side-chains, but most lack specificity and thus will modify all side-chains in addition to those of interest for example at the active site
• Example - Alkylation or acylation of ionizable R groups.

• Affinity labels build in selectivity by being targeted for the active-site through their structural resemblance to the substrate
• Majority of amino acid side-chains (reactive ones) are nucleophiles.
• Thus one will want to use modifiers which are electrophiles, i.e. species which will be susceptible to attack by nucleophiles.
• Note that a key effect with an affinity label is that it initially forms a non-covalent, EI, complex prior to undergoing a reaction typically with an nucleophile.
• There are also photoaffinity labels, typically diazo or azide compounds which are stable until flash-photolyzed and then generate very reactive species such as carbenes and nitrenes (R(C==O)CHN2 R(C==O)CH: or R--N3 R--N: + N2)

• Mechanism-Based inhibition involves generation of the reactive form of the inhibitor during the reaction, i. e., the enzyme initially recognizes the inhibitor as a substrate, starts interacting with it, and thereby generates a species which typically forms a stable covalent bond to the enzyme, thus irreversibly blocking the active site
• Example = FdUMP and thymidylate synthase

• Alteration of the number of enzyme molecules
• Alteration of enzyme activity (see p. 182-183)
• Control by low MW species
• Control by enzyme modification
• Compartmentalization

The rate of synthesis is mediated by a variety of mechanisms at both the transcriptional (e.g. repression, induction, derepression) and translational levels.

Summary of Methods for regulation Enzyme Levels

• Turnover
• A function of the rate of the proteins synthesis and degradation rates
• Synthesis is regulated at several levels
• Transcription - rate of synthesis of the mRNA(s) encoding the protein(s)
• Translation - rate of synthesis of the protein(s) form the mRNA(s) and its assembly into appropriate conformation
• Degradation: control of enzyme degradation is function of
• age (protein half-life: time a protein typically is stable in a cell)
• structural integrity
• activity of degradative enzymes

• Control by low molecular weight species
• Substrate
• Concentration (in relation to velocity of reaction) relative to K_m
• Concentration relative to allosterism (also see PFK and ADP regulation)(remember hemoglobin positive cooperativity)

• Product
• Feedback inhibition -- biosynthesis of isoleucine (Fig. 8-2) or ATCase in pyrimidine biosyn. (Figs. 10-2 to 10-11)
• In a typical metabolic pathway,

A ----> B ----> C ----> D ----> E ----> F
  |
  |
 G----> H----> I

where A, B, C etc represent metabolites, and each arrow represents an enzyme.
Imagine also that branching occurs from C and E with an additional series of reactions

• Control of the system requires that if too much of either or both of the end products build up then the rate of formation of that compound must be decreased.
• Similarly if too much reactant (A) builds up then the rate of conversion of A to products should be increased.
• From the engineering point of view it makes sense to control the flux at the first step of the pathway and at each branch point.
• This is exactly what is found in nature. The ultimate products, which probably bear no resemblance to the substrate A, inhibit the enzymes at A--> B and at the branch points.
• By having two different enzymes which catalyze at A --> B, it is possible to exert finer control then with just one, each one being inhibited by one of the end metabolites but not the other.
• The first step in a metabolic pathway is usually called the "committed step".
• It is at these steps that the enzymes are subject to major control.
• Another good example illustrating this type of feedback inhibition is the regulation of aspartate metabolism in E. coli.
• Since the structures of the end-products of the metabolic pathways usually bear no structural resemblance to the substrates of the first step, how does the inhibition occur at the enzyme molecular level?
• Allosterism.

• Allosteric effectors = Allosterism
• For viability of the cell it is critical that the concentration of most metabolites be very closely controlled, i.e. there is normally a very close tolerance on the allowable metabolite concentration.
• To keep such tight control the enzymes controlling metabolite interconversion have to be very sensitive to the overall balance required to maintain the metabolic system.
• If you examine the Michaelis Menten equation you will find that an increase in v from 0.1 to 0.9 Vmax requires an 81-fold change in substrate concentration. In other words the velocity is rather insensitive to substrate concentration.

0.1Vmax = VmaxS1/( Km + S1)
0.1Km + 0.1S1 = S1 Km = similarly 0.9Vmax = VmaxS9/(Km + S9)
0.9 Km + 0.9 S9 = S9
0.9 Km = 0.1 S9 Km = 0.11
S9 0.11S9 = 9S1 and S9/S1 = 81

• Exactly the same factor applies for inhibitors. Thus nature has turned to "cooperative" systems, these are ones in which a small change in one parameter, e.g. inhibitor or activator concentration, brings about a large change in velocity.

• A consequence of a cooperative system is that the v vs. S plot is no longer hyperbolic but becomes sigmoidal (see Fig. 8-22 & 8-25)((see PFK ADP regulation or general cooperative regulation)
• This has the advantage that a small change in the concentration of the effector ligand (substrate, inhibitor, activator), will bring about a large change in the rate. In other words the system becomes like a switch.

• To understand allosterism one must understand that it is based on ligand interactions and conformational changes. There are a number of basic aspects to consider.
• Fundamentally the phenomenon usually involves two or more subunits which can exist in two or more different conformations, in which each different conformation has a different catalytic activity.
• Normally there will be two conformations, one very active and one inactive or relatively inactive. Binding of ligands (S, I, A) to the enzyme perturbs the position of the equilibrium between the two conformations.
• For example an inhibitor will favor the inactive form, an activator will favor the active form. A corollary of this is that there will be different binding sites for the effector ligands (I, A) and the substrate.
• See Stryer Fig. 8-23

• The cooperative aspect comes in as follows
• If we consider dimers, then a positive cooperative event is one in which binding of the first ligand makes binding of the next easier.
• Negative cooperativity means that the second ligand will bind less readily then the first.

• Allosteric regulation
• Allosteric effectors can +/- alter enzyme action by binding to allosteric site
• This form of regulation is a common way to respond to changing conditions within a cell
• Example = surplus/deficit of ATP, AA's, metabolic intermediates
• Intermediate enzymes are not required (allows for faster responses to changes in cellular environment)
• Allosteric enzymes usually occupy key positions in metabolic pathways
• first unique step in a pathway
• first step of a branch pathway
• often regulated by ATP, ADP, AMP or Pi (reflect the energetic state of the cell)
• Phosphofructokinase example (see reaction and ADP regulation)
• committed step in glycolysis - first enzyme unique to pathway; major enzyme regulated in pathway
• catalyzes the transfer of Pi from ATP to the OH on carbon 1 of fructose-6-phosphate
• ATP acts as substrate and allosteric effector (see Figure alone or with other figures)
• negative effector - above 1 mM
• positive effector - below 1 mM
• other allosteric effectors - ADP (+), AMP (+), GDP (+),

cAMP (+), F-1,2-bisP (+), PEP (-) and citrate (-)
• PEP feedback to regulate entire glycolytic pathway

• Control by enzyme modification
• Covalent modification
• Phosphorylation - addition/removal of PO₄ to the OH- containing sidechains of Ser, Tyr, or Thr via kinases (requires ATP or GTP)
• Often a response to hormonal signals (extracellular signals)
• Can affect sensitivity of allosteric enzyme to +/- effectors, thus altering regulation
• Phospho form is active and non-phosphorylated form is inactive
• Will discuss Glycogen phosphorylase later in course
• Enzymes which alter phosphorylation state
• kinases - add PO₄ (transferases)
• phosphatases - remove PO₄ (hydrolases)

• Adenylation - addition/removal of AMP to Y

• Protein processing into active form such as through proteolytic processing is common form of activation of proteases (see Fig. 8-4)
• Digestive proteases are produced as inactive forms called zymogens or pro-enzymes:
• Trypsinogen is synthesized in the pancreas and activated by proteolytic cleavage in the small intestine
• Proteolytic processing ranges from removal of dipeptide (actually two) in chymotrypsin, to as much as close to 200 residues in -lytic protease.
• The activation of inactive proteases is also critical in the amplification in blood clotting, and is also used in other systems.
• For more supplemental information on this topic
• See proteases and blood-coagulation (serine proteases in cascade for activation of thrombin)

• Non-covalent modification
• Protein-protein interactions of regulatory proteins
• Calmodulin regulation of enzymes by Ca++ (see Fig. 8-3)
• calmodulin is a protein subunit associated with many enzymes
• modifies the activity of enzymes containing calmodulin as intracellular concentration of CA++ ions change
• small protein - mw 17,000, two globular domains connected by alpha-helix
• conformation of the protein changes when Ca binds; change is translated into enzyme activation/inactivation

• Structural alterations to E which affects either K_m or k_cat
• Conformational alterations
• Through reversible binding of an effector - affects either K_m or k_cat
• Allosteric effectors bind to an allosteric site of an enzyme
• Usually small molecules - ATP, but may be other proteins

• Generally have a more complex structure than unregulated enzymes
• Typically oligomeric molecules that have separate binding sites for substrates and modulators (allosterics)

Isozymes are similar enzymes encoded by distinct genes which carry out a similar reaction but have slightly different kinetic and allosteric properties. They provide for specific and fine regulation in a cellular compartment or cell type.

• Hexokinase represents an interesting example.
• The enzyme catalyzes the phosphorylation of glucose to glucose-6-phosphate. (Kinases are enzymes which transfer phosphate groups).
• Brain requires almost all its energy in the form of glucose, whereas other tissues can utilize fats and amino acids.
• The brain hexokinase has a low Km value for glucose, 0.05 mM.
• It is thus able to phosphorylate glucose and make it available for brain metabolism, even when the tissue or blood levels of glucose are low, e.g. starvation or fasting.
• This is critical to brain function and survival
• The liver isozyme, which is called glucokinase, has a much higher Km, 10 mM, and reaches its maximal activity only when blood glucose levels are about twice normal (5.5 mM), as after a meal.
• This is because its primary role in the liver is to remove excess blood glucose for storage as glycogen, an energy reserve.

• In this case control is exerted by two enzymes with widely differing Km's, hence being effective under quite different conditions = isoforms or isozymes.

---

---

---

COURSE HOMEWORK PROBLEMS and STUDY AID:

Study Questions & Answers  on Bioenergetics and Kinetics

---

COURSE STUDY AID and LEARNING OBJECTIVES:

Learning objective questions on kinetics

Learning objective questions on enzyme regulation and mechanisms 

---

---

OnLine Supplement:

Problems & Answers on Enzyme Biochemistry (Introductory Course in Biology at MIT):

Solving Chemical Equilibrium Problems

Solving Enzyme Mechanism Problems

3.5 Solving Enzyme Kinetics Problems

Solving Feedback Regulation Problems

Practice Problems!

---

For an advanced tutorial on enzyme kinetics:

Enzyme Kinetics Tutorial at Jefferson

---

---

 Lecture 11 - 3521 

---

---

BACK to the Top of this document