Chapter 6- Enzymes'

What are Enzymes

Enzymes are Catalysts- increase reaction rates without being used up

Most enzymes are globular proteins but some are RNA

The study of enzymatic processes is the oldest field of biochemisty- late 1700

The study of ezymes has dominated biochemistry in the past and continues to do so

Biocatalysis vs Inorganic Catalysts

Greater reaction specificity: Avoid side products

MIlder Reaction Conditions: Conductive to conditiond in cells (pH 7, 37C)

Higher Reactions Rates: in biologically useful timeframe

Capasity for regulation: Control of biological pathways

• Metabolites have many potential pathways of decompositiion

• Enzymes make the desired product the most favorable

How to Lower Delta G

Uncatalyzed biomolecular reactions

• Two free reactants go to a single restricted transition state- conversion is entropically unfavorable

Uncatylyzed Unimolecular reactions

• Flexible reactant goes to ridig transition state

• - transition is entropically unfavorable for flexible reactants

Catalyzed reactions

• The enzyme uses the binding energy of substrates to organize the reactats to a fairly rigid ES complex

• The entropy cost is paid during binding

• Rigid reactant complex goes to transition state and conversion is entropically neutral

Transition States

Transition state is the point right before the substrate turns into products

Linus Pauling- Enzymes bind transition states best

• enzyme active sites are complimentary to the transition state of the reaction

• enzymes bind transition states better than substrates

• stronger/additional interactions with the transition state as compared with the ground state lower the activation barrier

What is Enzyme Kinetics

Kinetics is the study of the rate at which compounds react

Affected by:

• Enzyme Concentraction

• Substrate concentration

• Effectors ( examples- Inhibitors and magnessium)

• Temperature

Michaelis Mention Equation

Assumptions:

• [ES] is constant - formation = breakdown

• [P] is negligent at Vo

• [S] is much greater from [E]

kcat

Turnover Number

How many subtrate molecules one enzyme molecule can convert per second

Km

Michaelis Constant

An approximate measure of a substrates affinity for an enzyme

Lower number when more affinity

Vmax

During steady state occurs when all of the enzyme is in the Es complex and is dependent on the breakdown of that complex

Lineweaver-Burk Plot

Linearized plot that is good for analysis of two-substrate data or inhibition

a non linear MM plot should be used to calculate parameters for Km and Vmax

Specificity

kcat / Km

diffusion from the active site limits the maximum values for specificity

can gain efficiency by having a high velocity or affinity for substrate

2-Substrate Reactions

Kinetic mechanism: the order of binding of substrates and release of preoducts

Sequencial vs Ping Pong

Sequencial Kinetic Mechanism Graph

Cannot easily distinguish random from ordered

Normally Random mechanisms will give the intersection point at the y-axis

Lineweaver-Burk Plot: Lines intersect

Ping-Pong Kinetic Mechanism Graph

Lineweaver-Burk: Lines are parallel

Enzyme Inhibition

Inhibitors are compounds that decrease an enzymes activity

Irreversible inhibitors react with the enzyme

• one inhibitor molecule can permanently shit off one enzyme molecule

• they are often powerful toxins but also may be used as drugs

Reversible inhibitors bind to and can dissociate from the enzyme

• they are oten structural analogs of substrates or products

• they are often used as drusg to slow down a specific enxyme

• Can bind to the free enzyme and prevent binidng of the substrate or bind to the ES complex and prevent the reaction

Competitive Inhibition

Competes with substrate for binding

• binds with the active site

• does not effect catalysis

No change in the Vmax

Apparent increase in Km

LWB: Lines intersect at the y-axis

Uncompetitive Inhibition

Only binds to ES complex

• does not effect substrate binding

• inhibits catalytic function

Decrease in Vmax

Apparent decrease in Km

No change in Vmax / Km

LWB: lines are parallel to each other

Mixed Inhibition

Binds with enzyme with or without substrate

• binds to regulatory site

• inhibits both substrate binding and catalysis

Decrease in Vmax

Change in Km

LWB: lines intersect at a point left of the y-axis

Noncompetitive are mixed inhibitors but with no change in Km

Noncovalent Modification

Allosteric Regulators

are generally small chemicals

can be positive or improve ezymatic catalysis

can be negative and reduce enzymatic catalysis

Enzyme Activity Regulation

Noncovalent Modification- Allosteric

Irreversible Covalent Modification- Zymogens

Reversible Covalent Modification- Phosphorylation Adenylylaiton

Catalytic Mechanisms

Enzymes may us one or more of the following

• Acid-base catalysis: give and take protons

• Covalent catalysis: change reaction paths

• metal ion catalysis: use redox cofactors and pKa shifters

Amino Acids in Acid-Base Chemistry

Glu/Asp ; COOH to COO-

Tyr/Ser ; OH to O-

Lys/Arg ; NH3+ to NH2

His ; NH+ to N

Cys: SH to S-

Nucleophile

Donated a pair of electrons to form a covalent bond

Example: O- to OH

Electrophiles

Accpets an electron pair to form a colalent bond

Chymotrypsin

Needs Asp, His, and Ser - called proteases catalytic triad

Peptidoglycan and Lysozyme

petidoglycan is a polysaccharide found in many bacterial cell walls

Clevage of the cell wall leads to the lysis of bacteria

Lysozyme is an antibacterial enzyme

Peptidoglycan and lysozyme Mechanism

Asp acts as a nucleophile to attack the anomeric carbon in the first SN2 Step

Glu acts as a Gerneral Acid and protonates the leaving group in the transition state

Water Hydrolyzes the covalent glycosyl-enzyme intermediate

Glu 35 acts as a general base to deprotonate water is the second SN2 Step

Sn1 vs Sn2

SN1:

• Two Steps- leaving group leaves then nucleophile attacks

SN2:

• One step- Nucleophile attacks and leaving group attacks at the same time

Cross Linking Peptidoglycan

Polysaccharides and peptides cross-linked via transpeptidase reaction

Beta Lactamase

When Beta- lactamase bind to penicillin it causes the penicillin to become inactive by adding an oxygen

When Beta Lactamase binds to Clavulanic acid BL gets inactivated

Cofactors

Molecules that bind to an enzyme and are required for catalytic activity

• Metals- Fe, Mn, Co, Cu, Zn, Mo

• Coenzymes- small organic molecules that are often derived from vitamins

  • May loosly bind and be released

  • May bind tightly / permanetly (prostetic groups)

Apoenzymes vs Holoenzymes

Apo- ezymes that do not have required cofactors ( can work without one)

Holo- enzymes that have a required cofactor (Need it to work)