Metabolic Pathways & Enzyme Regulation

I. Overview of metabolism

Metabolism is the sum total of all of the enzymatically catalyzed reactions occurring in the cell.

-specific functions

-Metabolism is divided into two phases

catabolism

anabolism

What is Intermediary Metabolism?

II. The Stages of Metabolism

The complex made simpler!

The three stages of catabolism

The degradation of large macromolecules proceeds through a number of consecutive molecular reactions organized into three major stages:

stage 1

stage 2

stage 3

The three stages of anabolism

example: protein synthesis

stage 3

stage 2

stage 1

Other Features of Anabolic and Catabolic Pathways

1. convergent and divergent

2. not the reverse

3. different locations

III. Nature of Enzyme Catalysis of Metabolism

The product of one reaction becomes the substrate of the next reaction

"multienzyme system"

Three levels of complexity:

a. simple systems

b. multienzyme complex (pyruvate dehydrogenase reaction complex)

c. organized multienzyme complex (non cyclic photophosphorylation)

IV. Metabolic Regulation

Introduction

Regulation: why do you need it?

1. Regulation by mass action

• dG = dGº + RTlnKeq
• What does K_eq stand for?
  
• The overall K_eq of a metabolic pathway is what's important
  

2. Regulation by enzyme activity

The key to metabolic regulation

A. Altering enzyme activity

1. substrate concentration

M-M curve

What about in the cell (in vivo)?

the importance of S concentrations near the Km

2. K_m for substrate

Consider the following hypothetical pathway:

          C

          ­   enzyme “C” (K_m for substrate B = 5 mM)

A ® B

          ¯   enzyme “D” (K_m for substrate B = 50 mM)

          D

What does the Km tell you about enzyme-substrate affinity?

Consider the shape of the M-M curve and return to the hypothetical branch point

Is the path to "C" is always favored because of the lower K_m for substrate of the enzyme which catalyzes that reaction?

3. Isozymes

What are they?

example: lactate dehydrogenase

catalyzes the following reaction:

pyruvate + NADH ---> lactate + NAD

5 different forms found in vertebrates that are combinations of two polypeptide chains "M" and "H": thus, we have M₄, M₃H, M₂H₂, MH₃, H₄ isozymes

M₄ is mostly found in muscle tissue and has a low K_m and high V_max

H₄ is mostly found in heart tissue and has a high K_m and low V_max

Explain the differences in kinetic constants for each isozyme. Hint: what are some differences between heart muscle and skeletal muscle?

4. Allosteric effectors

Allosteric effector binding site

What happens when an effector is bound to the allosteric site?

What sorts of molecules can be allosteric effectors?

Mechanism for allosteric regulation

example: phosphofructokinase

an enzyme from glycolysis; reaction catalyzed:

fructose-6-P <---> fructose-1,6-diP

ADP & GDP (what is GDP?) are allosteric activators; phosphoenolpyruvate is an allosteric inhibitor (this is an end-product of the pathway)

(Remember previous discussion regarding the ATP cycle and enzyme regulation of ATP generating and ATP producing reactions?)

X-ray diffraction studies were done of the enzyme in active "R" and inactive "T" states

Thus, T--> R requires ADP or GDP

How have the chemical properties of the active site changed in the shift from "T" to "R"?

Types of allosteric interactions

a. feedback inhibition

b. feedback stimulation

c. feedforward stimulation

another example of feedback inhibition:

aspartate transcarbamoylase (ATC ase): 12 polypeptide chains

reaction

this is the first step in the biosynthesis of CTP and UTP

CTP acts as a negative allosteric effector of ATCase

ATP acts as a positive allosteric effector of ATCase

Look at the kinetic curve for ATCase with or without ATP and CTP.

What happens to the K_m and V_max values for aspartate under each condition?

How does this data help to show the nature of the allosteric effect for each modulator?

5. Covalent modification of enzyme activity

a. enzymes are sometimes produced in an inactive form called a zymogen

example:

pepsinogen--> pepsin + peptides (42 amino acids)

b. enzymes are modified by kinases and phosphatases

B. Regulating the Number of Enzyme Molecules

A complex topic not extensively covered in this cell biology course (details to be found in genetics and molecular biology)

The number of enzyme molecules in the cell is a function of the regulation of the genes that code for those enzymes.

Three components of gene control

1. Signals

What are the signals to which a specific gene responds?

2. Levels

At which level does regulation occur?

3. Mechanisms

How is transcription regulated? Translation?

How do hormones affect protein synthesis?

How are parts of the DNA made available for transcription?