Day 3 - Enzyme kinetics, allosteric enzymes, covalent modifications

Km

The substrate concentration at which the speed of the reaction is exactly half the max velocity

Km inversely reflects

Enzyme affinity

Decrease in Km = increase in enzyme affinity - not much substrate needed

Increase in Km = decrease in enzyme affinity - need lots of substrate

Activator

Increases enzyme affinity which decreases Km

Competitive inhibition in regards to Km

Decreases enzyme affinity which increases Km

Increase Vmax by

Induction, increase gene expression of enzyme

Upregulation of receptors or transporters

Decrease Vmax by

Repression, silencing gene expression

Downregulation of receptors or transporters

Noncompetititve inhibition

Competitive Inhibitors do what

Compete with the substrate for binding at the active site of the enzyme

Are competitive inhibitors structurally similar to substrate

Yes

Competitive inhibitors and the active site

Block the enzymes active site

Can competitive inhibitors dissociate from active site?

Yes

Competitive inhibitors and substrate concentration

Increases substrate concentration

Competitive Inhibitors and reversibility

Reversible with increased substrate concentration

Competitive Inhibitors Km and Vmax

Km increased

No change in Vmax

3 examples of competitive inhibitors

Allopurinol for gout

Lovastatin for reducing cholesterol

5-flurouracil for cancer

Noncompetitive Inhibitors do what

Bind to the enzyme or the enzyme-substrate complex at a site different from the active site

Noncompetitive Inhibitors: structurally similar to substrate?

No

Noncompetitive Inhibitors and the active site

Alters the conformation of the enzyme along with the active site

Noncompetitive Inhibitors: remains bound to enzyme until

the unavailability of the substrate

Noncompetitive Inhibitors: substrate concentration

Does not change substrate concentration

Noncompetitive Inhibitors: reversibility

Generally irreversible

Noncompetitive Inhibitors: Km and Vmax

No change in Km

Vmax decreased

3 Examples of non-competitive inhibitors

Cyanide which stops ATP production

Fluoride which alters glycolysis

Dimercaprol which alters cell respiration

Allosteric Inhibition of Enzyme Activity

Binding to allosteric site changes enzyme active site so substrate can’t bind

Graph for allosteric enzymes is

sigmoidal

Allosteric Activation of Enzyme Activity

Binding to allosteric site changes conformation of active site so that substrate can bind

Activators of Allosteric Enzymes

Bind substrate more readily

Shift the kinetic curve to the left, decreasing Km

Inhibitors of Allosteric Enzymes

Bind substrate less readily

Shift the curve to the right, increasing Km

Isozymes vary regulation

Vary regulation of the same reaction at distinct locations or times

Isozymes are

Homologous enzymes that catalyze the same reaction but differ slightly in structure and Km, Vmax values

Examples of Isozymes

Hexokinase-1 and glucokinase

Hexokinase = found in erthyrocytes, skeletal muscles, and others

Glucokinase = low-affinity enzyme found in liver

Phosphorylation: Donor molecule, Group added, Function

Donor = ATP

Group = Phosphate

Function = glucose homeostasis and energy production; signal transduction

Protein kinases

Add phosphate

Phosphatases

Remove phosphate

AAs involved in phosphorylation

Serine, tryosine, threonine

(Histidine less frequently)

Adenylation: Donor molecule, Group added, Function

Donor = ATP

Group = AMP

Function = enzymatic activity, co-factor binding

AAs involved in adenylation

Tyrosine and threonine

Serine less frequently

ADP-Ribosylation: Donor molecule, group added, function

Donor = NAD

Group = ADP ribose

Function = cell signaling, DNA repair, gene regulation, cell death, immune response

Methylation: Donor molecule, Group added, Function

Donor = SAM

Group = methyl

Function = cell signaling, epigenetic regulation

Cholera toxin causes

Gsa-Arg-ADP-ribosylation

Diphtheria toxin causes

EF2-diphthamide-ADP-ribosylation

Pertussius toxin causes

Gia-Cys-ADP-ribosylation

Botulinum C3 protein ADP-ribosylates

GTP-binding proteins Rho and Ras

Covalent Modifications (4)

Phosphorylation

Adenylation

ADP-Ribosylation

Methylation