Lecture 7 & 8 (first half)

Multi-substrate enzyme interactions

ternary complex

• random: all substrates bind to enzyme in random order forming the ternary complex

• ordered: substrates bind in an ordered fashion, 1 after the other

Ping-pong mechanism

• subtrate 1 binds and releases product forming an enzyme intermediate for substrate B to pick up and form a different product

• no ternary complex formed

• ex: hexokinase (ATP and Glucose)

each substrate has a Km

Types of reversible inhibition

competitive

uncompetitive

non competitive (mixed)

competitive inhibition

inhibitor competes with ONLY active site → NO catalysis

• forms E1 complex

• Km INCREASES, Vmax CONSTANT

alphaKm is the Km observed with comp. inhibitor is present

uncompetitive inhibition

inhibitor binds in spot OTHER than active site (allosteric site)

• conformational change renders enzyme INACTIVE

• forms ES1 complex

• Km DECREASES, Vmax DECREASES

alpha’Km is the Km observed when uncomp. inhibitor is present

mixed inhibition

inhibitor binds in spot OTHER than active site

• either E or ES complex (has bias toward one)

• Km INCREASES, Vmax DECREASES

• kinetics impacted by alpha and alpha ‘

non-competitive inhibition

a special case of MIXED inhibition

alpha = alpha’

can bind either E or ES complex

• Km CONSTANT, Vmax DECREASES

irreversible inhibition

suicide inhibitors - form covalent bonds with their targets

• structurally similar to the normal substrate so the enzyme treats it like a substrate catalyzing the mechanism

• permanently bonded to the active site

• forms a reactive intermediate covalently traps a catalytics residue in the active site

ex: penicilin acts as a suicide inhibitor of bacterial glycopeptide transpeptidase

Regulatory enzyme function

enzymes positioned at key points in metabolic pathways that regulate catalytic activity in response to cellular signals

does not obey michaelis-menten kinetics

Feedback inhibition

regulatory mechanism where the end product of a multi-step metabolic pathways acts as a negative allosteric modulator (inhibitor) of the first enzyme in the pathway

• saves cellular resources and energy

allosteric modulation

when binding of a molecule on one part of a protein affects the shape and behavior of the protein

have an active site and regulatory site (BOTH specific)

• positive modulators: stabilize the active site, high affinity R state, lowers the K0.5, INCREASES catalytic rate

• negative modulators: stabilize the T state, raises K0.5 and DECREASES catalytic activity

heterotropic modulator

enzymes are when the modulator is different from substrate binds outside active site

• positive modulators: stabilize the active site, high affinity R state, lowers the K0.5, INCREASES catalytic rate (can shift curve to left - hyperbolic like)

• negative modulators: stabilize the T state, raises K0.5 and DECREASES catalytic activity (can shift the curve to the right)

homotropic modulator

enzymes are when the substrate is the modulator (active site is regulatory site)

Types of reversible covalent modifications

methylation: addition of methyl group

ADP-ribosylation: addition of ribose

acetylation: addition of acetyl group

adenylation: addition of an AMP to tyrosine

myristoylation:

Prenylation: addition of lipid

ubiquitination: addition of ubiquitin to lysine

phosphorylation: addition of phosphate groups

importance of phosphorylation

thermodynamically favorable & rapid

• kinase: adds a phosphoryl group to a protein (takes it off ATP first) (recognize specific sequences)

• phosphatase: removes a phosphoryl group from an enzyme

neg charge introduced electrostatic interactions or steric clashes that either active or inactive an enzyme

forms the basis of kinase cascades that relay extracellular info throughout the cell

Amino Acid targets of phosphorylation

all have OHs (nucleophilic)

Serine

Thereonine

Tyrosine

Low Activity → High Activity

• 2 ATP → 2 ADP

• 2 OH Phosphorylase b → 2 P Phosphorylase a

High Activity → Low Activity

• 2 H2O → PP1 → 2 Pi

• 2 P Phosphorylase → 2 OH Phosphorylase b

Low Blood sugar → High Blood sugar

• glucagon

• ATP → PKA → ADP

• phosphorylase kinase (P_

High Blood sugar → low blood sugar

• insulin

• H2O → PP1 → Pi

• phosphorylase kinase (OH)

irreversible activation

occurs by proteolytic cleavage of a zymogen (precursor)

cleavage triggots a localized conformational rearrangement opening up a properly aligned catalytics residues → converts zymogen into fully active enzyme

trypsin: a protease that is activated by proteolytic cleavage - breaks down proteins in the gut

• zymogen: trypsinogen

Proteases

peptidases are enzymes that catalyze the hydrolysis of peptide bonds linking amino acids in proteins

uses:

• nutritional digestion (pepsin, trypsin)

• blood coagulation (fibrin)

• programmed cell death

• protein turnover

classes:

• serine

• cysteine

• aspartyl (HIV- protease)

• metallo-

Serine Proteases Mechanism (Chymotrypsin, Trypsin)

1. substrate binding: polypeptide chain is positioned near active site with the polypeptide chain near serine

2. nucleophilic attack (acylation): catalytic triad (Ser-His-Asp) polarizes serine. serine OH attacks the carbonyl carbon of the peptide bond, forming a high energy TETRAHEDRAL intermediate (stabilized by oxyanion hole)

3. bond cleavage & amine release: tetrahedral intermediate collapses, breaking the peptide bond, N-term departs forming acyl-enzyme intermediate

4. deacylation: catalytic water molecule enters active site and nucleophilically attacks the acyl-enzyme bond with His stabilization

5. product: second tetrahedral intermediate collapses, releasing the c-term peptide and regenerating serine

Beta-lactamases

1. nucleophilic attack: serine OH attacks the carbonyl of the strained Beta-lactam ring of penicillin

2. Beta-lactam ring opens covalently forming a stable acyl-enzyme intermediate trapping the enzyme

3. hydrolysis: beta-lactamase hydrolyzes the covalent bond with the addition of catalytic water resulting in an opened, inactive antibiotic molecule

Chymotrypsin vs Trypsin

substrate specificity pockets differ

• chymotrypsin: deep, hydrophobic substarte pocket lined with non polar amino acids (Gly & Ala)

  • accommodates and binds bulky, hydrophobic aromatic amino acid side chains (Phe, Tyr, Trp)

• Trypsin: neg charged (asp)

  • attracts and forms strong salt bridges with pos charged basic side chains (Lys, Arg)

AA based on pH vs Activity curves

rising phase: catalytic residue must be deprotonated (inflection point on this slope corresponds to pka)

falling phase: second catalytic must be protonated (inflection point on this slope gives pka)

at high pH: salt bridge btw alpha amino group and Asp is broken → closes active site

low pH: his is protonated, cannot interact in catalysis