Copyright © 2000-2014 Mark Brandt, Ph.D. 55 Regulation of Enzymes Control of any metabolic process depends on control of the enzymes responsible for mediating the reactions involved in the pathway. Because regulating metabolic pathways is critically important for living organisms, the ability to regulate enzymatic activities is required for survival. A number of methods are used to regulate enzymes and the rates of the reactions they catalyze. Control of the amount of enzyme Two methods can be used to change the amount of an enzyme present in a cell: 1) change the rate of enzyme synthesis 2) change the rate of enzyme degradation The effect of both of these processes is to change the net amount of enzyme. Because Vmax is directly proportional to the enzyme concentration, and because the velocity of a reaction is directly proportional to the Vmax, changing the amount of enzyme changes the rate of a reaction. A number of hormones induce changes in cellular functioning by altering the enzyme concentration. Changing the amount of an enzyme is conceptually simple method for changing the amount of enzyme activity. However, altering enzyme concentration is a relatively slow process (the minimum time required is about 15 minutes to allow increased or decreased protein synthesis to have an effect). As a result, other methods are frequently used in addition to effects related to altered gene expression. Control of the type of enzyme In many cases, more than one form of an enzyme will catalyze a particular reaction. Different isoenzymes or isozymes are products of different genes. Some multimeric proteins can be synthesized from more than one isozyme. The resulting multimers are different isoforms. In most cases, the different isozymes have somewhat different properties, and therefore can be used to regulate the reaction, or the rate of the reaction, that occurs. Different isozymes may have markedly different affinities for the same substrate. An example of this is provided by hexokinase and glucokinase, which both catalyze the phosphorylation of glucose. Although they catalyze the same reaction, these enzymes differ dramatically in OHOOOHOHCH2HOHOHOOOHOHCH2OHPOOOATP ADPHexokinaseGlucokinaseCopyright © 2000-2014 Mark Brandt, Ph.D. 56 their affinity for glucose. The normal concentration for glucose in the bloodstream varies from 4 to 6.5 mM depending on conditions (glucose concentration rarely varies outside this range in normal individuals, although abnormal states such as diabetes mellitus may result in much larger variation in glucose concentration). The graph indicates that hexokinase will be nearly saturated with substrate over the entire normal range. However, glucokinase activity will increase significantly as glucose concentration rises from 4 to 6.5 mM. These enzymes are present in different tissues. Hexokinase is expressed in tissues where glucose utilization is regulated by other processes, and it is necessary for hexokinase to operate at maximal rate regardless of the glucose concentration. In contrast, glucokinase is found largely in the liver and pancreas, where the ability of glucokinase to increase its velocity with increasing glucose concentration has an important regulatory role for glucose metabolism. We will see these enzymes again during the section on carbohydrate metabolism. Control of preformed enzyme In addition to modulating the amount of an enzyme, it is possible to modulate the activity of an enzyme. These alterations in activity can involve changes in Km or kcat or both. Possible mechanisms include: 1) Covalent modification of the enzyme (most commonly by phosphorylation). 2) Protein-protein interaction. For example, calmodulin binds calcium; when bound to calcium calmodulin binds to a number of other proteins, and regulates their activity. 3) Competitive inhibition by substrate analogs 4) Non-competitive inhibition by small molecules 5) Allosteric effectors (see below) With the exception of alterations in activity mediated by competitive inhibitors, these mechanisms for control of preformed enzyme all involve changes in the conformation of the protein, induced either by the covalent or non-covalent binding of the modulator molecule. 00.20.40.60.810 2 4 6 8 10 12!"#$%&'()*+,-./(0&1234'(*+5!*6*789:*,-."#$%&1234'(*+5"#$*6*7:8*,-.%;<!&'Copyright © 2000-2014 Mark Brandt, Ph.D. 57 Allostery and Cooperativity Allostery Allosteric means “other shape” or “other space”. The term refers to phenomena where ligand binding at one site affects a different, distant site, and to the fact that this binding alters the shape of the protein. Allosteric effects can occur within one subunit and hence, do not necessarily require an oligomeric protein structure or an enzyme that displays cooperativity relative to the substrate. Allosteric effectors that decrease activity are termed negative effectors, whereas those that increase enzyme activity are called positive effectors. When the substrate itself serves as an effector, the effect is said to be homotropic. In such a case, the presence of a substrate molecule at one site on the enzyme alters the catalytic properties of the other substrate-binding site. Homotropic effects can occur in proteins with more than one subunit: if each subunit contains a catalytic site for the same enzymatic process, the binding of substrate at one site may alter the affinity or catalytic rate at other active sites in the complex. Homotropic effects can also involve interactions between regulatory sites (with no catalytic function), and active sites (with no regulatory function). Alternatively, the effector may be different from the substrate, in which case the effect is said to be heterotropic. Heterotropic effectors tend to bind to allosteric sites; binding of heterotropic effectors to the active site is unusual. Effector binding can alter either the Km or the Vmax of the enzyme. Effectors that alter the Vmax are termed V-type effectors. Effectors that alter the Km are called K-type effectors. Most (although not all) K-type effectors modulate the activity as a result of