enzymes kinetics, inhibition, regulation muhammad jawad hassan assistant professor biochemistry

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ENZYMES KINETICS, INHIBITION, REGULATION Muhammad Jawad Hassan Assistant Professor Biochemistry

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ENZYMES KINETICS, INHIBITION, REGULATION

Muhammad Jawad HassanAssistant Professor

Biochemistry

Michaelis-Menten kinetics

Vmax approachedasymptotically

V0 = Vmax x[S]/([S] + Km)

V0 is moles of productformed per sec. when [P]is low (close to zero time)

Michaelis-Menten Equation

E + SESE + P

Michaelis-Menten Model

V0 varies with [S]

Steady-state & pre-steady-state conditions

At equilibrium, no net change of [S] & [P]or of [ES] & [E]

At pre-steady-state,[P] is low (close to zerotime), hence, V0 for initial reaction velocity

At pre-steady state, we can ignore the back reactions

Michaelis-Menten kinetics (summary)Enzyme kinetics (Michaelis-Menten Graph) :

At fixed concentration of enzyme, V0 is almost linearly proportionalto [S] when [S] is small, but is nearly independent of [S] when [S]is large

Proposed Model: E + S ES E + P k2

ES complex is a necessary intermediate

Objective: find an expression that relates rate of catalysis to the concentrations of S & E, and the rates of individual steps

k1

Michaelis-Menten kinetics (summary)

Start with: V0 = k2[ES], and derive,

V0 = Vmax x[S]/([S] + Km)

At low [S] ([S] < Km), V0 = (Vmax/Km)[S]

At high [S] ([S] > Km), V0 = Vmax

When [S] = Km, V0 = Vmax/2. Thus, Km = substrate concentration at which the reaction rate (V0) is half max.

Range of Km values

Km provides approximation of [S] in vivo for many enzymes

Lineweaver-Burk plot (double-reciprocal)

Allosteric enzyme kinetics

Sigmoidal dependence of V0 on [S], not Michaelis-Menten

Enzymes have multiple subunitsand multiple active sites

Substrate binding may be cooperative

Enzyme inhibition

A competitive inhibitor

MethotrexateA competitive inhibitor of dihydrofolate reductase - role in purine& pyrimidine biosynthesis

Used to treat cancer

Kinetics of competitive inhibitor

Increase [S] toovercomeinhibition

Vmax attainable,Km is increased

Ki =dissociationconstant forinhibitor

Competitive inhibitorVmax unaltered, Km increased

Kinetics of non-competitive inhibitor

Increasing [S] cannotovercome inhibition

Less E available,Vmax is lower,Km remains the samefor available E

Noncompetitive inhibitorKm unaltered, Vmax decreased

Enzyme inhibition by DIPFGroup - specific reagents react with R groups of amino acids

diisopropylphosphofluoridate

DIPF (nerve gas) reacts with Ser in acetylcholinesterase

Affinity inhibitor: covalent modification

Catalytic strategies commonly employed

1.Covalent catalysis. The active site contains a reactive group, usually a nucleophile that becomes temporarily covalently modified in the course of catalysis

2. General acid-base catalysis. A chemical reaction is catalyzed by an acid or a base. The acid is often the proton and the base is often a hydroxyl ion. A molecule other than H2O may play the role of a proton donor or acceptor.

3. Metal ion catalysis. Metal ion can function in several ways;

• can serve as an electrophile, stabilizing a negative charge on a reaction intermediate. • can generate a nucleophile by increasing the acidity of a nearby molecule, such as H2O in the hydration of CO2 by carbonic anhydrase. • can bind to substrate, increasing the number of interactions with the enzyme.

4. Catalysis by approximation. Bringing two substrates together along a single binding surface on an enzyme

Enzyme specificity: chymotrypsinCleaves proteins on carboxyl side of aromatic, or large hydrophobic amino acid

Bonds cleaved, indicated in red

The enzyme needs to generate a powerful nucleophile to cleave the bond

A highly reactive serine (#195) in chymotrypsin

27 other serines not reactive to DIPF,Ser 195 is a powerful nucleophile

DIPF: di-isopropylphosphofluoridate, only reacts with Ser 195

Covalent catalysis

Hydrolysis in two stages

Acylation to form acyl-enzyme intermediate

Deacylation to regeneratefree enzyme

Ser 195 OH groupattacks the carbonyl group

Acyl-enzyme intermediateis hydrolysed

Chymotrypsin in 3D3 chains; orange,blue, & green

Catalytic triad ofresidues, includingSer 195

2 interstrand, &2 intrastranddisulfide bonds

See Structural Insights

Synthesized as chymotrypsinogenProteolytic cleavage to 3 chains

The catalytic triad (constellation of residues)

Ser 195 converted into a potent nucleophile, an alkoxide ion

Imidazole N asbase catalyst,accepts H ion,positions &polarizes Ser

Asp 102orientsHis 57

H ion withdrawal from Ser 195 generatesalkoxide ion

1. Allosteric control. Proteins contain distinct regulatory sites and multiple functional sites. Binding of regulatory molecules triggers conformational changes that affect the active sites.

Display cooperativity: small [S] changes - major activity changes. Information transducers: signal changes activity or information shared by sites

2. Multiple forms of enzymes (isozymes). Used at distinct locations or times. Differ slightly in structure, in Km & Vmax values, and in regulatory properties

3. Reversible covalent modification. Activities altered by covalent attachment of modifying group, mostly a phosphoryl group

4. Protleolytic activation. Irreversible conversion of an inactive form (zymogen) to an active enzyme

Regulatory Strategies: Enzymes & Hemoglobin

Aspartate transcarbamoylase reaction

Committed step in pyrimidine synthesis: inhibited by end productCTP

CTP inhibits ATCase

CTP stabilizes the T state

CTP binds toregulatorysubunits

R and T states in equilibrium

ATCase displays sigmoidal kineticsSubstrate binding to one active site converts enzyme to R stateincreasing their activity: active sites show cooperativity

Basis of sigmoidal curveR & T states equivalent to 2 enzymes with different Kms

Cooperativity

Effect of CTP on ATCase kineticsCTP stabilizes the T state, curve shifts to right

Effect of ATP on ATCase kineticsATP, allosteric activator, stabilizes R state, curve shifts to left

Oxygen delivery by hemoglobin, cooperativity enhanced

Partial pressure of oxygen

98 - 32 = 66%

63 - 25 = 38%

Cooperativityenhances delivery 1.7 fold

Heme group structure

4 linked pyrrole ringsform a tetrapyrrolering with a centraliron atom.side chains attached

Position of iron in deoxyhemoglobin

Iron slightly outsideporphyrin plane

His (imidazole ring)binds 5thcoordination site

6th site for O2 binding

O2 binding, conformational change

Iron moves into plane, his is pulled along

Quaternary structure of hemoglobin

Pair of identicalalpha-beta dimers

Transition from T-to-R state in hemoglobin

As O2 binds, top pair rotate 15o with respect to bottom pair

Interface most affected

Oxygen affinity of fetal v maternal red blood cells

Fetal Hgl does notbind 2,3-BPG,higher O2 affinity

Fetal hemoglobintetramer has2 alpha & 2 gamachains,

Gene duplication

Isozymes of lactate dehydrogenase: glucose metabolism

Rat heart LDH isozyme profile changes with development

H(heart) isozyme (chain)= square, M(muscle) isozyme = circle

Tissue content of LDHFunctional LDH is tetrameric, with different combinations of subunits possible. H4 (heart) has higher affinity for substrates than does M4 isozyme, different allosteric inhibition by pyruvate

H4

H3M

H2M2

HM3

M4Some isozymes in blood indicative of tissue damage, used for clinical diagnosisIncrease in serum levels of H4 relative to H3M, indicative of myocardial infraction (heart attack)

Examples of covalent modification

Phosphorylation widely used for regulation

Gammaphosphorylgroup

Some known protein kinases

Protein phosphotases

Reverse the effects of kinases, catalyze hydrolytic removal ofphosphoryl groups attached to proteins

Activation by proteolytic cleavage

Secretion of zymogens by acinar cell of pancreas

Pancreas, one of the mostactive organs insynthesizing & secretingproteins

Acinar cell stimulated byhormonal signal ornerve impulse, granulecontent released intoduct to duodenum

Proteolytic activation of chymotrypsinogen

Active enzyme generatedby cleavage of a singlespecific peptide bond

3 chains linked by 2interchain disulfidebonds, (A-B & B-C)

Conformations of chymotrypsinogen & chymotrypsin

Electrostatic interactionbetween Asp 194carboxylate & Ile 16-amino group possibleonly in chymotrypsin,

essential for activity

Zymogen activation by proteolytic cleavage

Digestive proteins of duodenum

Secreted by cells that line duodenum

Zymogens orange, active enzymes yellow

Interaction of trypsin with its inhibitor

Lys 15 & Asp 189form salt bridgeinside the activesite

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