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What are three basic ways cells use to regulate metabolites through its enzyme-catalyzed pathways in a way which is responsive to changes in its surroundings?

1. By changing substrate concentration or availability.
2. By changing the relative activity or efficiency of enzymes.
3. By changing the quantity of enzyme present. The regulation of gene expression is very important here. An extreme example of this in higher organisms is differentiation--the behavior of cells as liver, brain, or skin cells even though they all contain the same genetic makeup.


How does substrate concentration or availability affect cell regulation?

A. For many enzymes the Km or affinity for a substrate is close to the physiological substrate concentration. Under these conditions the rate of substrate utilization is responsive to changes in its concentration.
B. The effective metabolite concentration in the immediate neighborhood of the enzyme is important here, not the average concentration throughout the cell.
C. The availability of metal ions and cofactors may also be critical for enzyme activity.
D. Enzymes are often compartmentalized. That is, they are found only in certain specific organelles and not equally distributed throughout the cell. For example, the respiration chain is located in the mitochondria. The physical separation of a metabolic process facilitates its regulation independent of processes in the rest of the cell.
E. Sometimes enzymes which catalyze a sequence of reactions are organized as a macromolecular complex. The metabolites can be passed along from one enzyme in the complex to the next, so to speak. For example, fatty acid synthetase is a large, multienzyme complex.


What are the two major ways to alter enzyme activity?

Allosteric regulation and covalent modification


Describe how allosteric regulation affects enzyme activity.

The catalytic activity of regulatory enzymes is modulated by allosteric effectors which usually have no structural similarity to the substrate. The effector binds to a regulatory site (or allosteric site) on the enzyme, causing a conformational change which affects the catalytic activity of the enzyme. Allosteric inhibitors decrease enzyme activity. Allosteric activators increase enzyme activity. Feedback inhibition refers to the inhibition of an enzyme early in a biosynthetic pathway by an end product of that pathway. Feedback inhibitors are also called negative allosteric effectors.
For a sequence of enzyme reactions catalyzing the steps in a metabolic pathway, one of the steps will be the "slowest". This rate-limiting step limits the velocity of the overall pathway. A change in the velocity of the rate-limiting step effects the flux through the whole pathway. The committed step is an essentially irreversible step which is unique to that pathway. Once material passes through the committed step, it is committed to the rest of the pathway. Frequently the committed step and the rate-1imiting step are the same. The enzyme catalyzing this step is usually controlled by products of the pathway.


How does covalent modification work?

i) "Interconvertible" enzymes exist in 2 forms--active or inactive--depending upon whether or not the enzyme has been covalently modified by the attachment of some type of group
to it. In mammals a phosphate group is usually attached. In bacteria a nucleotide is often used. For some enzymes the phosphorylated form is active. In others the phosphorylated form is inactive. The phosphorylation and dephosphorylation are catalyzed by specific "converter" enzymes. This type of regulation is often under hormonal control.

ii) Activation by proteolysis:
Some enzymes and hormones are initially synthesized in an inactive form. Protease
action removes part of the molecule to produce the active form.
e.g. proinsulin > insulin trypsinogen > trypsin
Chymotrypsinogen > chymotrypsin


Explain gene regulation in general and how it is used to control enzyme concentration?

A basic mechanism of regulation of an enzymic process is by altering the quantity of enzyme present. This is usually accomplished by regulation of some point in the expression of the
gene which codes for that enzyme.
The fact that regulation of gene expression does in fact occur can be illustrated by the situation in E. coli. If all genes were expressed at the same rate, then one would expect all proteins
to be made in equal amounts. However, the number of copies of different proteins actually varies over a very wide range. For example, in each E. coli cell there are about 15,000 copies of each ribosomal protein, 100,000 copies of each enzyme in the glycolytic pathway, and about 5 copies of # -galactosidase. Furthermore, some enzymes are made in fixed, constant amounts (e.g. enzymes of the glycolytic pathway), while others can very dramatically in concentration depending on the availability of certain nutrients in the environment (e.g. #-galactosidase).
In higher organisms, gene regulation is also used in the process of differentiation -- the formation of different cell types from single parental cells.


T or F. In order to allow for the most efficient utilization of nutrients, bacteria tend to make onlythose enzymes that are immediately necessary for them.



What are Constitutive enzymes and what do they do in bacterial gene expression?

Constitutive enzymes are made in fixed amounts. They are required by the bacteria in fairly constant amounts at all times. An example would be the enzymes of the glycolytic pathway. This type of enzyme is sometimes referred to as a "housekeeping" enzyme.
-non-regulated, always present


Describe inductive enzymes.

Another class of enzymes is useful for the catabolism or utilization of certain nutrients only when those nutrients are present in the growth medium. These enzymes are made at only a low basal level in the absence of the particular nutrient, but in its presence the synthesis of the enzymes is induced. For example, the enzyme Beta-galactosidase is made only when lactose is present in the growth medium.


Describe repression-depression enzymes.

A third class of enzymes is used for the biosynthesis of metabolites (e.g. amino acids).
These enzymes are made when the end product is not present in the growth medium. However, if the end product is available from the medium the synthesis of these enzymes is repressed (i.e. they are not made). An example would be the enzymes involved in tryptophan biosynthesis.

(It should be obvious from these 3 examples that bacteria regulate enzyme levels by controlling the rate of synthesis of the enzymes. This is usually accomplished by regulating the rate
of transcription.)


What type of enzyme is the lac operon of e.coli?



Describe the lac operon system.

The system consists of three enzymes: beta-galactosidase, galactoside permease, and galactoside acetylase. When E. coli is growing in medium containing glucose these enzymes are only present at a low level. When glucose + lactose is present the enzymes are still at a low level. When only lactose is present the enzymes are produced at a high level. The enzymes undergo coordinate expression--that is, they are all expressed in about equal amounts (either all high or all low). The structural genes (i.e. the genes coding for the amino acid sequence of the enzymes) are physically associated with regulatory genes to form the operon.
(Pic slide 6)


Describe the lac operon system in with only glucose present in the medium.

The repressor binds to the operator to block transcription. This represents negative control.
(Low cAMP)


Lac operon system in presence of only lactose?

The inducer binds to the repressor to release it from the operator. This allows the RNA polymerase to proceed. Since glucose is low, the cAMP is high. cAMP binds to the CAP protein which in turn binds to the CAP binding site. This helps the RNA polymerase to effectively bind to the promoter. This is positive control. The three enzymes are all produced from one long polycistronic message

(cAMP high)


Describe lac operon system in presence of glucose and lactose.

The inducer releases the repressor from the operator. However, cAMP is low, since glucose is high, so that no cAMP-CAP complex is present to allow RNA polymerase to bind. This effect of glucose is called "catabolite repression

(cAMP low)


Describe the Jacob and Monod Model for enzyme repression.

The Jacob and Monod Model for Enzyme Repression
The idea of the operon as a controlling unit is attributed to Francois Jacob and Jacques Monod. Their hypothesis can be used to form a model for enzyme repression as well as induction. Regulation by enzyme repression and derepression is usually used in anabolic pathways - that is, the biosynthesis of some end product. Generally, if the end product is available from the medium, then the enzymes of the biosynthetic pathway are not made. If the end product is absent from the medium, then the enzymes are made.
-The tryptophan biosynthetic pathway is regulated by this mechanism.
-In this model, the repressor has no affinity for the operator unless it is complexed with the corepressor (the end product). Thus, if the end product is not available from the medium, then the repressor does not bind to the operator and the enzymes are all coordinately derepressed. They are all made from a polycistronic message. If the end product is available in the medium, then the repressor-corepressor complex is formed. This complex binds to the operator and blocks transcription by inhibiting the progress of the RNA polymerase which binds at the promoter site.
-(basically negative feedback)


What is attenuation?

Several prokaryotic operons involved in amino acid biosynthesis (thus, repressible operons) are regulated by premature termination of mRNA synthesis, a process called attenuation.
-In high amino acid concentrations, mRNA synthesis is terminated prematurely so that the biosynthetic enzymes are not made. In low amino acid concentrations, mRNA synthesis is completed so that the biosynthetic enzymes are made (coordinate expression). Whether the mRNA transcript is completed or terminated prematurely depends on which of several possible secondary structures form at the 5' end of the nascent mRNA. The decision as to which secondary structure can form is dependent on the rate of movement of the first ribosome to translate the message.


How does amino acid concentration affect synthesis of mRNA, related to attenuation? Example given is with Histidine.

The 5' ends of the mRNA's from these operons typically encode a short (probably nonfunctional) polypeptide called the “leader peptide” that contains several codons for the amino acid whose synthesis is controlled by the operon. If the amino acid is plentiful in the cell (i.e. the biosynthetic enzymes are not needed), ribosomes translate this region quickly, which favors formation of an RNA secondary structure (“attenuator stem”) that leads to rho-independent termination of transcription. If the amino acid is depleted in the cell (i.e. the biosynthetic enzymes are needed), the ribosomes stall during translation of this segment, which favors formation of an RNA secondary structure that allows completion of the mRNA transcript (usually a “polycistronic” message). Similar models have been developed for regulation of the histidine, leucine, tryptophan, phenylalanine, and threonine operons. As an example, consider the regulation of the histidine operon of Salmonella.


What's an example of where specific chromosomes or chromosomal regions may be inactivated during development?
(Possible control point)

One X chromosome in each female mammalian cell is inactivated by condensation to heterochromatin (Lyon hypothesis).


What's an example where specific chromosomal regions may be activated by translocation?
(Possible control point)

Genes for the variable and constant regions of antibody molecules may be joined by translocation before they are activated.


What is an example where Specific chromosomal material may be lost during development? (specific point of control)

The human red blood cell loses its entire nucleus.


What's an example of Gene reiteration - the presence of many identical genes permits the corresponding transcripts to be made at a high rate? (Possible control point)

Xenopus has about 900 rRNA genes in each cell.