REGULATION OF ENZYME ACTIVITY Medical Biochemistry, Lecture 25
Lecture 25, Outline • General properties of enzyme regulation • Regulation of enzyme concentrations • Allosteric enzymes and feedback inhibition • Other effectors of catalytic activity
General Properties: Regulatory Enzymes • The biochemical pathways that you will soon be studying are composed of groups of coordinated enzymes that perform a specific metabolic process. In general, these enzyme groups are composed of many enzymes, only a few of which are regulated by the mechanisms described in this lecture. Regulatory enzymes are usually the enzymes that are the rate-limiting, or committed step, in a pathway, meaning that after this step a particular reaction pathway will go to completion.
General Properties: Regulatory Enzymes (cont) • Frequently, regulatory enzymes are at or near the initial steps in a pathway, or part of a branch point or cross-over point between pathways (where a metabolite can be potentially converted into several products in different pathways). In general, a cell needs to conserve energy - therefore costly (in metabolic terms) biosynthetic reaction pathways will not be operational unless a particular metabolite is required at a given time.
General Properties: Regulatory Enzymes (cont) • Recall that when acting as catalysts, enzyme mediated-reactions should be reversible. However, regulatory enzymes frequently catalyze thermodynamically irreversible reactions, that is, a large negative free energy change (-DG) greatly favors formation of a given metabolic product rather than the reverse reaction. Thus, regulation of enzyme activity, usually at the committed step of the pathway, is critical for supplying and maintaining cellular metabolitic and energy homeostasis.
Two General Mechanisms that Affect Enzyme Activity: • 1) control of the overall quantities of enzyme or concentration of substrates present • 2) alteration of the catalytic efficiency of the enzyme
Regulation of Enzyme Concentrations • The overall synthesis and degradation of a particular enzyme, also termed its turnover number, is one way of regulating the quantity of an enzyme. The amount of an enzyme in a cell can be increased by increasing its rate of synthesis, decreasing the rate of its degradation, or both.
Regulation of Enzyme Concentrations: Induction • Induction (an increase caused by an effector molecule) of enzyme synthesis is a common mechanism - this can manifest itself at the level of gene expression, RNA translation, and post-translational modifications. The actions of many hormones and/or growth factors on cells will ultimately lead to an increase in the expression and translation of "new" enzymes not present prior to the signal. These generalizations will be covered in more detail in Dr. Bannon's lectures.
Regulation of Enzyme Concentrations: Degradation • The degradation of proteins is constantly occuring in the cell, yet the molecular mechanisms that determine when and which enzymes will be degraded are poorly understood. The turnover number of an enzyme can be used for general comparison with other enzymes or other enzyme systems, yet these numbers can vary from minutes to hours to days for different enzymes.
Regulation of Enzyme Concentrations: Degradation (cont) • Protein degradation by proteases is compartmentalized in the cell in the lysosome (which is generally non-specific), or in macromolecular complexes termed proteasomes. Degradation by proteasomes is regulated by a complex pathway involving transfer of a 76 aa polypeptide, ubiquitin, to targeted proteins. Ubiquination of protein targets it for degradation by the proteasome. This pathway is highly conserved in eukaryotes, but still poorly understood
Regulation of Enzyme Concentrations: Degradation (cont) • Proteolytic degradation is an irreversible mechanism. For examples, rapid proteolytic degradation of enzymes that were activated in response to some stimulus (for example, in a signal transduction response). This type of down-regulation allows for a transient response to a stimulus instead of a continual response. Establishing the links between proteasomes, ubiquination and signal transduction pathways is currently a very active research area
Zymogens: Inactive Precursor Proteins • A clinically important mechanism of controlling enzyme activity is the case of protease enzymes involved (predominantly) in food digestion and blood clotting. Protease enzymes (enzymes that degrade proteins) like pepsin, trypsin and chymotrypsin are synthesized first as larger, inactive precursor proteins termed zymogens (specifically pepsinogen, trypsinogen, and chymotrypsinogen, respectively).
Zymogen Protease Examples Chymotrypsinogen cleavage sites to yield active chymotrypsin
Zymogens (cont) • Activation of zymogens by proteolytic cleavage result in irreversible activation. Zymogen forms allow proteins to be transported or stored in inactive forms that can be readily converted to active forms in response to some type of cellular signal. Thus they represent a mechanism whereby the levels of an enzyme/protein can be rapidly increased (post-translationally). Other examples of zymogens include proinsulin, procollagen and many blood clotting enzymes (the latter will be discussed in the next lecture).
Enzyme/Substrate Compartmentation • Segregation of metabolic processes into distinct subcellular locations like the cytosol or specialized organelles (nucleus, endoplasmic reticulum, Golgi apparatus, lysosomes, mitochondria, etc.) is another form of regulation. Enzymes associated with a given pathway frequently form organized, multi-component macromolecular complexes that perform a particular cellular process. Similarly, it follows that the substrates associated with a given pathway can also be localized to the same organelle or cytosolic location. This segregation allows for more specialized regulation of cellular processes.
Allosteric Enzymes • Allosteric enzymes - from the Greek allos for "other" and stereos for "shape" (or site) meaning "other site". These enzymes function through reversible, non-covalent binding of a regulatory metabolite at a site other than the catalytic, active site. When bound, these metabolites do not participate in catalysis directly, but lead to conformational changes in one part of an enzyme that then affect the overall conformation of the active site (causing an increase or decrease in activity, hence these metabolites are termed allosteric activators or allosteric inhibitors).
Allosteric Example • Feedback Inhibition - This occurs when an end-product of a pathway accumulates as the metabolic demand for it declines. This end-product in turn binds to the regulatory enzyme at the start of the pathway and decreases its activity - the greater the end-product levels the greater the inhibition of enzyme activity. This can either effect the Km or Vmax of the enzyme reaction.
Allosteric Enzymes - Properties • Allosteric enzymes differ from other enzymes in that they are generally larger in mass and are composed of multiple subunits containing active sites and regulatory molecule binding sites. The same principles that govern binding of a substrate to an active site are similar for an allosteric regulator molecule binding to its regulatory site.
Kinetics of Allosteric Enzymes - Terms • Cooperativity - in relation to multiple subunit enzymes, changes in the conformation of one subunit leads to conformational changes in adjacent subunits. These changes occur at the tertiary and quaternary levels of protein organization and can be caused by an allosteric regulator. • Homotropic regulation - when binding of one molecule to a multi-subunit enzyme causes a conformational shift that affects the binding of the same molecule to another subunit of the enzyme. • Heterotropic regulation - when binding of one molecule to a multi-subunit enzyme affects the binding of a different molecule to this enzyme (Note: These terms are similar to those used for oxygen binding to hemoglobin)
Allosteric Enzymes - Kinetics • Allosteric enzymes do exhibit saturation kinetics at high [S], but they have a characteristic sigmoidal saturation curve rather than hyperbolic curve when vo is plotted versus [S] (analogous to the oxygen saturation curves of myoglobin vs. hemoglobin). Addition of an allosteric activator (+) tends to shift the curve to a more hyperbolic profile (more like Michaelis-Menten curves), while an allosteric inhibitor (-) will result in more pronounced sigmoidal curves. The sigmoidicity is thought to result from the cooperativity of structural changes between enzyme subunits (again similar to oxygen binding to hemoglobin). NOTE: A true Km cannot be determined for allosteric enzymes, so a comparative constant like S0.5 or K0.5 is used.
Regulation by Modulator Proteins - Calmodulin Calmodulin is a small protein (17 kDa) that can bind up to four calcium ions (blue dots) in the two globular domains. When calciumis bound, calmodulin acts as a protein co-factor to stimulate the activity of target regulatory kinases like phosphorylase kinase, myosin kinase, Ca-ATPase and a Ca/calmodulin-dependent protein kinase. It is the structural conformation of Ca-calmodulin that makes it an active co-factor
Regulation of Enzyme Activity by Covalent Modifications • Another common regulatory mechanism is the reversible covalent modification of an enzyme. Phosphorylation, whereby a phosphate is transferred from an activated donor (usually ATP) to an amino acid on the regulatory enyme, is the most common example of this type of regulation. Frequently this phosphorylation occurs in response to some stimulus (like a hormone or growth factor) that will either activate or inactivate target enzymes via changes in Km or kcat.
Phosphorylation/Signal Transduction • Phosphorylation of one enzyme can lead to phosphorylation of a different enzyme which in turn acts on another enzyme, and so on. An example of this type of phosphorylation cascade is the response of a cell to cyclic AMP and its effect on glycogen metabolism. Use of a phosphorylation cascade allows a cell to respond to a signal at the cell surface and transmit the effects of that signal to intracellular enzymes (usually within the cytosol and nucleus) that modify a cellular process. This process is generically referred to as being part of a signal transduction mechanism
Signaling Regulation of Glycogen Synthase and Phosphorylase A-forms, most activeB-forms, less active
Other covalent modificiations: • Prenylation, Myristoylation, Palmitoylation: The covalent addition of hydrophobic, acyl fatty acid or isoprenoid groups to soluble proteins/enzymes can alter their intracellular location. This type of hydrophobic acylation generally causes target proteins to associate with a membrane rather than the cytosol. Thus, it represents a mechanistic and functional re-compartmentalization of the target protein/enzyme (an example of a prenylated protein is the Ras oncogene discussed in lecture 11)
Allosteric and Phosphorylation Regulation - Glycogen Phosphorylase