B2 mechanisms of enzyme catalysis Flashcards by lily stratton

what is the essence of catalysis

the specific stabilisation of the transition state

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what do catalysts do

-lower the activation energy
-accelerator of a chemical reaction
-increase the rate of rxn
-is not consumed in the rxn
-does not affect the eqm

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how is the active site of an enzyme formed

folding of protein brings side chains of various aa that may be far apart in the primary seq into close juxtaposition

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how many steps are in the enzyme-catalysed reaction

3
enz+S– enz-S–enz-P–enz+p

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what allows the reaction to occur in regards to active site and substrate

positioning of the substrate mols in the most favourable relative orientation for the rxn to occur

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the active site is perfectly ??? to the transition state

complementary

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what do the aa side chains of the active site do to the electron distribution of the transition state

stabilise

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the substrate is ??? on binding to the active site

strained

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enzymes do what to the activation energy and reaction rate

lowers the activation energy
increases the reaction rate

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what are the non covalent interactions between the substrate and the aa side chains of the enzyme

-acidic groups (Asp, Glu)= ionic bonds
-basic groups (Lys, His, Arg)= ionic bonds
-hydrophillic ints woth -OH or alc groups (Ser, Thr, Tyr)
-hydrophilic ints with -SH or thiol groups (Cys)
-hydrophilic ints with amide groups (Asm, Gln)
-aromatic ints (Phe, Tyr, Trp)
-hydrophobic ints (Ala, Leu, Lle, Val, Met)

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how do reactive groups at the catalytic site surface catalyse the rxn by

-donating or withdrawing electrons
-stabilising or generating free radical intermediates
-forming temporary covalent bonds
(transition state intermediate)

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what are cofactors

non protein molecules in addition to enzymes

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3 examples of cofactors

-metal group (hexokinase Mg2+)
-prosthetic group
covalently bound organic mol (heme, lipoic acid)
-coenzyme
tightly but not covalently bound organic mol (NAD)

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name of an enzyme protein WITH prosthetic group/coenzyme and is it catalytically active

holo-enzyme
catalytically active

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name of an enzyme protein WITHOUT prosthetic group/coenzyme and is it catalytically active

apoenzyme
catalytically inactive

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what does specificity mean

enzymes catalyse only one specific rxn

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oxidoreductases (general)

oxidation and reduction reactions

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dehydrogenases

addition or removal of H

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oxidases

2 electron transfer of O2 forming H2O2
2 electron transer to 1/2 O2 forming H2O

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oxygenases

incorporate O2 into product

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hydroxlases

incorporate 1/2 O2 into product as -OH and form H2O

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peroxidases

use as H2O2 as oxygen donor, forming H2O

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transferases (general)

transfer a chemical group from one substrate to another

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kinases

transfer phosphate from ATP onto substrate

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hydrolases

hydrolysis of C-O, C-N, O-P, and C-S bonds

examples of hydrolases

esterases, proteases, phosphatases, thioesterases

lyases

addition across a carbon-carbon double bond

examples of lyases

dehydratases, hydratases, decarboxylases

isomerases

intramolecular rearrangements

synthetases

formation of bonds between two substrates frequently linked to utilisation of ATP

units of catalytic/enzyme activity

number of micromoles (umol) of substrate converted per minute under standard optimised conditions at 30 degrees 1 enzyme unit (EU)= 1umol min-1

what is specific activity

activity of an enzyme per milligram (mg) of total protein in the enzyme prep (expressed in umol min-1mg-1)

what does specific activity give a measurement of

purity of the enzyme

why is the rxn rate hyperbolic

accumulation of product depletion of substrate denaturation of enzyme

requirements of measuring the rxn rate

measured at fixed enzyme conc defined temp and pH

for meaningful quantitative assays of enzyme activity it is necessary to ensure what

that initial velocities (V0; rxn rates) are measured

5 factors that affect enzyme activity

-pH -temperature -concentration of enzyme -concentration of substrate -covalent modification of enzyme

what is pH and its parameters

a measure of the acidity of alkalinity of a solution acidic < 7.00 < basic neutral pH = 7.00

what of the aa side chains depends on the pH of the sol

ionisation state

what is dependent on the pH of an enzyme catalysed rxn

binding of the substrate and catalysis

do chemical reactions proceed faster or slower at higher temps and why

faster mols move faster, greater chance to strike electrons gain activation energy easier

what happens when an enzyme is denatured

loss of H bonding unfolding precipitation loss of activity

the effect of varying the amount of enzyme

predictable linear increase in product formation with increasing amount of enzyme

what does the michaelis-menten eqn describe

the dependence of rate of rxn on concentration of substrate at steady state (ES formation balanced by its removal) and vast molar excess of substrate over enzyme [S]>>[E]

what do the following in the michaelis menten eqn mean v Vmax [S] Km

v rate of rxn Vmax maximal rate of rxn [S] conc of substrate Km Michaelis constant

what is the michaelis menten eqn

v= Vmax[S]/Km + [S]

what does high Km and low Kn correspond to in terms of substrate

high = low affinity low = high affinity

enzymes with low Km compared with the conc of substrate [S] in the cell act at their ?

max rate

do modest changed in the conc of substrate [S] have an effect on the rate of rxn

effect of rate of rxn where enzymes with a high Km and a small change in [S] conc

large change

experimental determination of Km and Vmax

-incubation of the enzyme under optimal conditions for a short time (assuming no change on conc of [S] or [product] is negligible compared with [S] -using range of [S] concs -plotting double reciprocal graph of rxn rate over [S] conc -extrapolating back from experimental points to determine intercepts

what is the plot given to the double reciprocal plot

lineweaver-burk 1/rate 1/Vmax -1/Km 1/[substrate]

what occurs in a sequential rxn (enzyme with 2 substrates)

each substrate binds in turn

what is a ternary complex and what do the lines look like on LB graph

complex containing three diff mols A-Enz-B converging

dihydrofolate reductase enzyme pathway

dihydrofolate+NADPH+H+----tetrahydrofolate+NADP+

ping pong rxn (enzymes with 2 substrates) and what do the lines look like on LB graph

one substrate reacts, and modifies enzyme then second substrate reacts with modified enzyme parallel lines

what is an allosteric enzyme and where are the often found

contain binding sites other "allo" than substrate binding sites often in multi subunit complex more than one active site in complex

what does the binding of a substrate to an allosteric enzyme lead to

binding to active site of first subunit leads to change in conformation facilitating binding of substrate to the other active site

what is a reversible inhibitor

non covalent (eqm) binding to enzyme many are relatively unspecific

mechanism of a reversible inhibitor

blocking substrate binding or hindering catalytic steps

what are irreversible inhibitors

inactivators bind to enzyme covalently (suicide inhibitor) many are substrate analogues undergo part of rxn transition state covalent intermediate does not break down

what is a competitive inhibitor

competes with substrate for binding at the active site is a function of the relative affinities of the substrate and the inhibitor for binding the enzyme inhibition is a function of relative conc of substrate and inhibitor

effect of competitive reversible inhibition on michaelis menten and LB

Vmax is unchanged, Km increased if enough substrate added, can overcome inhibitor

what are mixed inhibitors

they do not bind to the active site inhibitor can bind prior to substrate or to the enzyme-substrate complex

mixed inhibitors distort the substrate binding site which affects

-apparent substrate affinity -catalytic turnover (slowing catalysis)

effect of mixed inhibitors on Km and Vmax

either increase or decrease Km decrease Vmax

binding position and competition of non competitive inhibitors

binds to the enzyme at a position separate from the active site no competition for binding with the substrate

what happens to the affinity and rate of rxn with non competitive inhibitors

apparent affinity for the substrate is unchanged, but the rate of rxn is slowed

effect of non competitive inhibition on MM eqn and effect of adding more [S]

Km is unchanged, Vmax is decreased adding more substrate has no effect on the rate of rxn

effect of allosteric inhibitors on Km and apparent affinity of enzyme

increase the Km and lower to apparent affinity of enzyme for its substrate