Quiz #4/5

Quiz #4/5• #4: Glycolysis (Tuesday, Feb 20th)

• #5: TCA cycle (Monday, Mar 5th)

• Pathways are in the books

• Quiz will have the entire pathway:–All cofactors will be present–Random intermediate and enzymes removed

• You fill in the missing names

–Draw the structure for 1 intermediate• Indicated by a larger box

Enzyme Regulation

Conditions Affecting Enzyme Activity

pHtemperature

pH

Effects of pH on Enzyme Activity

• Protonation state of side chains

– Variation in protein structure

– Substrate binding

– catalysis

• Ionization of substrate

– Substrate binding

Temperature

RelativeActivity

ba

Temperature

Protein unfolding

Control of Enzyme Availability

Principles of Genetic Regulation

Types of Enzymes“Control of Gene Expression”

• Constitutive Enzymes: e.g. glycolytic enzymes and gluconeogenic enzymes

• Inducible Enzymes: e.g. -galactosidase

• Repressible Enzymes: e.g. ten enzymes of histidine biosynthesis

Negative Regulators[Bind to operators or upstream repression

sequences (URS)]

O

O

–I nducer

I nducible

e.g. lactose operonRegulator

(Repressor)Complex

–Corepressor

Regulator(Aporepressor)

Complex(Repressor)

Repressible

e.g. trp operon

Positive Regulators[Bind to promoters, enhancers or upstream

activation sequences (UAS)]

O

O +I nducer

I nducible

e.g. cAMPRegulator Complex

"Activator"

+Corepressor

Regulator"Activator"

Complex

Repressible

e.g. nit-2

Regulation of Enzyme Catalytic Activity

Covalent ModificationAllosteric Enzymes

Principles Governing Controls of Enzyme Catalytic

Activity

• Regulatory Enzymes

– Enzyme catalyzing committed, rate-limiting step (often first step)

– Thermodynamically highly favorable reaction

• Outcomes of Regulation

– Feedback inhibition (fbi) of biosynthetic pathways

– Modulation of metabolic flux

Reversible Covalent Modification

Page 390

Protein Modification(Phosphorylation/Dephosphorylation)

Non-covalent Modification

Effectors or Ligands

Positive: activatorsNegative: inhibitors

Allosteric Enzymes(Modulation of Enzyme Catalytic

Activity)

• Substrate Binding

• Catalytic Rate

• Both

Allosteric (Regulatory) Enzymes

R (Active)(I nactive)

SubstratesActivators

I nhibitor

T

Homotropic Effects

VO

[S]

posit ive cooperativity

negative cooperativity

M—M (Simple Enzyme)

Heterotropic Effects

positive ef fector

negative ef fector

no ef fectorVo

[S]

Figure 12-16

Glycogen Phosphorylase

Regulation of Biosynthetic Pathways

Rationale for Regulation

CentralMetabolite

Product(e.g. Amino Acid)

Biosynthesis

Catabolism

Macromolecules

Nutrient

Efficiency and Flexibility

Biological Efficiency

• Biosynthesis– Synthesize precursors not available in diet– Cease synthesis when precursors become

available in diet (pre-existing enzymes)– Produce precursors and macromolecules at

appropriate rates

• Catabolism– Degrade most appropriate nutrients at

appropriate rates

Biological Flexibility

• Adaptaton to Dietary Changes– Need for biosynthetic products– Catabolism of new nutrients– Control of pre-existing enzymes

• Metabolic Flux– Rates of metabolism reflecting needs

for energy and macromolecular synthesis

Competing Reactions: Regulation

A

B C

Enzyme 1 Enzyme 2

Control Mechanisms

• Control of Enzyme Availability

– Induction/repression

• Control of Enzyme Activity

– Covalent/Non-covalent

• Control of Substrate Availability

Types of Regulation

• Specific: pathway’s substrate or product

• General: needs for C or N sources or growth rates (e.g. energy charge)

Signals Mediating Regulation

Availability ofSubstrates or Products

(Ligands)

Regulatory Proteins

Biosynthetic Pathways

CentralMetabolite

Product(Amino Acid)

ATP ADP + Pi

Simple Feedback Inhibition

X

ATP

CentralMetabolite

Product(Amino Acid)

ADP + Pi

Complex Feedback Inhibition

CentralMetabolite

Product 1

Product 2

XX

X

Mechanisms of Complex Feedback Inhibition

• Cumulative: sum of individual inhibitions

• Concerted: both end products required for inhibition

• Isoenzyme: two enzymes, each inhibitable by different end product

Cumulative Feedback Inhibition

A

GF

EDCBA

GF

EDCB

A

GF

EDCB

Concerted Feedback Inhibition

A

GF

EDCBA

GF

EDCB

A

GF

EDCB

Isozymes

A

GF

EDCBA

GF

EDCB

A

GF

EDCB

Modulation of Metabolic Flux

Energy Charge

Energy Charge(Daniel Atkinson)

Steady-State E.C. = 0.93

ATP, ADP and AMP = Regulatory Ligands

Energy Charge 12

2ATP + ADP ATP + ADP + AMP

=

Energy Charge

Anabolic pathways

(Biosynthesis)

• Require ATP

• Activated– High EC (ATP)

• Inhibited– Low EC (AMP)

Catabolic Pathways

(Degradation)

• Produce ATP

• Activated– Low EC (AMP)

• Inhibited– Hig EC (ATP)