Enzymes lectures 5-6

lineweaver burk plot

easier to define exact Km and Vmax

• takes the inverse of the axes (makes the plot linear)

slope on a lineweaver-burk plot

Km/Vmax

X intercept on Lineweaver-Burk plot

-1/Km

Is Km ideally supposed to be big or small?

small so we can reach Vmax quickly

Why does changing pH affect reaction rates?

change the charge of substrate—> changing how the substrates cant fit can interact with the active site

k_{cat}

number of subunits converted to product by an enzyme at max velocity

what determines the k_{cat} ?

how long it takes to get to transition state

ternary complex

all the substrates are present at once before product is made

• either ordered or random

ping-pong reaction

one substrate comes in—> modify the enzyme —> leaves —> another substrate comes in —> product is formed —> leaves

how does ordered binding to a ternary structure work?

each substrate binders one at a time then the products leave one at a time

how does substrate binding randomly to a ternary complex work?

either substrate binds first then the other binds —> either product leaves first then the other

substrates in Cleland nomenclature

A, B, C, D (first being A)

products in Cleland nomenclature

P, Q, S, T

bi bi reaction

two substrates come in and two products leave (ternary structure)

Cleland nomenclature of successive enzymes in a ping pong reaction

1st- E —> F —> G (always has to go back to original E)

what does this graph indicate and what type of graph is it?

the pathway includes a ternary complex

• lineweaver-burk plot

What does this graph indicate

ping pong reaction

why do we need to regulate enzymes

to conserve energy in the body

enzyme inhibitors

show or stop enzyme catalysis

irreversible inhibitors

permanently inactivate enzymes

• usually on active site

what’s irreversible inhibitors work (4)

• covalently bind to a functional group needed

• destroy functional group

• target protein for degradation

• cause enzymes to fall apart

suicide inhibitors

• type of permanent inhibitor

• bind to active site and start doing the chemical reaction

• hold enzyme in a permanent intermediate

transition state analogs

look like a substrate in its transition state but its in its ground state —> bind most tightly to enzyme

• makes really stable bonds (not covalent) with enzyme —> no reason to leave so stays permanently

reversible inhibitors

like protein ligands have an on/off rate (bind—> release)

K_{I}

reversible inhibitor binding rate

competitive inhibitors

looks like substrate and competes with the substrate

• enzyme bind —> [EI] complex—> inhibitor falls off

when are competitive inhibitors useful in medicine?

when they have a large \frac{1}{K_{I}} (Ki is small) —> means inhibitor rate is beating regular rate

how do competitive inhibitors change Km and Vmax?

• change Km but not Vmax

  • because changes how often E binds to S but not how many E

(apparent) \alpha K_{m}

how much the inhibitor is affecting K_{m}

• no inhibitor \alpha=1

what type of inhibition?

competitive inhibition

types of inhibition that happens at the active site

competitive and some irreversible

types of inhibitors that alter active site structure

mixed, noncompetitive, and uncompetitive

mixed inhibitor

• reversible

• binds to the allosteric site on either the E or ES

mixed inhibition on a Lineweaver-burk plot

different y-intercepts and different X-intercepts

what type of inhibition

mixed inhibition

noncompetitive inhibition

type of mixed inhibitor where I binds to E and ES equally

noncompetitive inhibition on Km and Vmax

affects Vmax but Km of unbound enzymes

blue

noncompetitive inhibition

blue

noncompetitive inhibition

uncompetitive inhibition

binds to ES on allosteric site and makes it so the ES can’t get to transition state

uncompetitive inhibition on Km and Vmax

Vmax decreases

Km decreases- takes smaller amounts of substrate to reach Vmax

what type of inhibition is this?

uncompetitive inhibition

how to alter activation site without inhibitors?

covalent modification of enzyme

how to covalently modify an enzyme?

• adding a phosphate by protein kinase

• taking away a phosphoryl group with phosphatases

• adding methyl or acetyl groups

what does adding and taking away phosphorly groups do to the active site?

change active site and whether a substrate can get into it and do its work

why is phosphorylation important in enzyme control?

it can fine tune the activation of an enzyme base on the specific processes in which enzymes can be phosphorylation (complex long cascade or one step)

proteolytic cleavages

permanently removes a part of the protein

zymogen

precursor to proteases that need their inhibitory part cut off

proproteins/proenzymes

the protein that need to have an inhibitory part cleaved off

feedback inhibition

when end product binds to allosteric site and creates a conformational change to stop the S for binding to the E

blue

positive allosteric effectors

pink

negative allosteric effectors