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8-27b). Allosteric enzymes therefore show different kinds of responses in theirsubstrate-activity curves because some have inhibitory modulators,some have activating modulators, and some have both.Two Models Explain the Kinetic Behaviorof Allosteric EnzymesThe sigmoidal dependence of Vo on [S] reflects subunit cooperativity,and has inspired two models to explain these cooperative interactions.In the first model (the symmetry model), proposed by JacquesMonod and colleagues in 1965, an allosteric enzyme can exist in onlytwo conformations, active and inactive (Fig. 8-28a).
All subunits are inthe active form or all are inactive. Every substrate molecule that bindsincreases the probability of a transition from the inactive to the activestate.In the second model (the sequential model) (Fig. 8-28b), proposedby Koshland in 1966, there are still two conformations, but subunitscan undergo the conformational change individually. Binding of substrate increases the probability of the conformational change.
A conformational change in one subunit makes a similar change in an adjacentsubunit, as well as the binding of a second substrate molecule, morelikely. There are more potential intermediate states in this model thanin the symmetry model. The two models are not mutually exclusive;the symmetry model may be viewed as the "all-or-none" limiting caseof the sequential model. The precise mechanism of allosteric interaction has not been established. Different allosteric enzymes may havedifferent mechanisms for cooperative interactions.Figure 8-28 Two general models for the interconversion of inactive and active forms of allostericenzymes. Four subunits are shown because themodel was originally proposed for the oxygen-carrying protein hemoglobin.
In the symmetry, or all-ornone, model (a) all the subunits are postulated tobe in the same conformation, either all Q ^ o w a^"finity or inactive) or all Q (high affinity or active).Depending on the equilibrium, Kl9 between Q andQ forms, the binding of one or more substrate (S)molecules will pull the equilibrium toward the Qform. Subunits with bound S are shaded.
A possiblepathway is given by the gray shading. In the sequential model (b) each individual subunit can bein either the Q o r EH f° rm - A very large number ofconformations is thus possible, but the shadedpathway (diagonal arrows) is the most probableroute.11111111 11ss111111ss(§X§)!sss11111111\ 11 11ss111111\ 11 I1111\11ssssssss(a)sss(b)233Chapter 8 EnzymesOther Mechanisms of Enzyme RegulationIn another important class of regulatory enzymes activity is modulatedby covalent modification of the enzyme molecule.
Modifying groupsinclude phosphate, adenosine monophosphate, uridine monophosphate, adenosine diphosphate ribose, and methyl groups. These aregenerally covalently linked to and removed from the regulatory enzyme by separate enzymes (some examples are given in Box 8-4).An important example of regulation by covalent modification isglycogen phosphorylase (MT 94,500) of muscle and liver (Chapter 14),which catalyzes the reaction(Glucose )„ + PiGlycogen> (glucose ) n _! + glucose-1-phosphateShortenedglycogenchainR groups of specificSer residuesThe glucose-1-phosphate so formed can then be broken down into lactate in muscle or converted to free glucose in the liver.
Glycogen phosphorylase occurs in two forms: the active form phosphorylase a and therelatively inactive form phosphorylase b (Fig. 8-29). Phosphorylase ahas two subunits, each with a specific Ser residue that is phosphorylated at its hydroxyl group. These serine phosphate residues are required for maximal activity of the enzyme.
The phosphate groups canbe hydrolytically removed from phosphorylase a by a separate enzymecalled phosphorylase phosphatase:Phosphorylase a + 2H2O(More active)2ATP + phosphorylase b(Less active)CH2Phosphorylase b(inactive)phosphorylase b + 2Pj(Less active)In this reaction phosphorylase a is converted into phosphorylase b bythe cleavage of two serine-phosphate covalent bonds.Phosphorylase b can in turn be reactivated—covalently transformed back into active phosphorylase a—by another enzyme, phosphorylase kinase, which catalyzes the transfer of phosphate groupsfrom ATP to the hydroxyl groups of the specific Ser residues in phosphorylase b:2ADP + phosphorylase a(More active)The breakdown of glycogen in skeletal muscles and the liver isregulated by variations in the ratio of the two forms of the enzyme.
Thea and b forms of phosphorylase differ in their quaternary structure;the active site undergoes changes in structure and, consequently,changes in catalytic activity as the two forms are interconverted.Some of the more complex regulatory enzymes are located at particularly crucial points in metabolism, so that they respond to multipleregulatory metabolites through both allosteric and covalent modification. Glycogen phosphorylase is an example. Although its primary regulation is through covalent modification, it is also modulated in anoncovalent, allosteric manner by AMP, which is an activator of phosphorylase b, and several other molecules that are inhibitors.Glutamine synthetase of E. coli, one of the most complex regulatory enzymes known, provides examples of regulation by allostery, reversible covalent modification, and regulating proteins.
It has at leasteight allosteric modulators. The glutamine synthetase system is described in more detail in Chapter 21.OHICH2OH'2P2ATPphosphorvlasrphosphatase* 2H9OO2ADPOCH9CH9Phosphorylase a(active)Figure 8—29 Regulation of glycogen phosphorylaseactivity by covalent modification. In the active formof the enzyme, phosphorylase a, specific Ser residues,one on each subunit, are in the phosphorylatedstate. Phosphorylase a is converted into phosphorylase b, which is relatively inactive, by enzymaticloss of these phosphate groups, promoted by phosphorylase phosphatase.
Phosphorylase b can be reactivated to form phosphorylase a by the action ofphosphorylase kinase.234Part II Structure and CatalysisBOX 8-4Regulation of Protein Activity by Reversible Covalent ModificationFigure 1 Some wellstudied examples ofenzyme modificationreactions.A variety of chemical groups are used in reversiblecovalent modification of regulatory proteins to produce activity changes (Fig. 1). An example of phosphorylation (glycogen phosphorylase) is given inthe text. An excellent example of methylation involves the methyl-accepting chemotaxis protein ofbacteria.
This protein is part of a system that permits a bacterium to swim toward an attractant insolution (such as a sugar) and away from repellentchemicals. The methylating agent is S-adenosylmethionine (adoMet), described in Chapter 17.ADP-ribosylation is an especially interesting reaction observed in only a few proteins. ADP-ribose isderived from nicotinamide adenine dinucleotide(see Fig. 12-41). This type of modification occursfor dinitrogenase reductase, resulting in the regulation of the important process of biological nitrogen fixation. In addition, both diphtheria toxin andcholera toxin are enzymes that catalyze the ADPribosylation (and inactivation) of key cellular enzymes or proteins.
Diphtheria toxin acts on andinhibits elongation factor 2, a protein involved inprotein biosynthesis. Cholera toxin acts on a specific G protein (Chapter 22) leading ultimately toseveral physiological responses including a massive loss of body fluids and sometimes death.Amino acid residues knownto accept covalent modificatiiCovalent modificationATP ADPEnzOTyr, Ser, Thr, His_> Enz—P—O"PhosphorylationO"ATPEnz-> Enz —P—O—CH2IAdenylylationITyrI Adenine/O.OH'OHUTPEnz-> Enz —P—O—CH2Tyrj Uridine6"UridylylationOHOHNAD nicotinamidevEnzADP-Ribosylation-^—^-OyOCH2—O—P—O—P—O—CH2i Adenine>EnzArg, Gin, Cys,diphthamide (a modified His)HOHOHOHOHS-adenosyl- S-adenosylmethionine homocysteineMethylationEnzEnz—CH3GluChymotrypsinogen(inactive)245Trypsinogen(inactive)167/^Val-(Asp) 4 -Lys1 15 16Arg He245Trypsin(active)7235245Val-(Asp).j-Lys-Ile77 -Chymotrypsin(active)Chapter 8 Enzymes245Figure 8-30 Activation of the zymogens of chymotrypsin and trypsin by proteolytic cleavage.
Thebars represent the primary sequence of the polypeptide chains. Amino acids at the termini of thepolypeptide fragments generated by cleavage areindicated below the bars. The numbers representthe positions of the amino acids in the primary sequence of chymotrypsinogen or trypsinogen. (Theamino-terminal amino acid is number 1.)HegThr 147 -Asn 1481 13 16Leu HeAa-Chymotrypsin(active)245146 149Tyr AlaActivation of an enzyme by proteolytic cleavage is a somewhat different type of regulatory mechanism. An inactive precursor of the enzyme, called a zymogen, is cleaved to form the active enzyme. Manyproteolytic enzymes (proteases) of the stomach and pancreas are regulated this way. Chymotrypsin and trypsin are initially synthesized aschymotrypsinogen and trypsinogen, respectively (Fig.
8-30). Specificcleavage causes conformational changes that expose the enzyme activesite. Because this type of activation is irreversible, other mechanismsare needed to inactivate these enzymes. Proteolytic enzymes are inactivated by inhibitor proteins that bind very tightly to the enzyme active site. Pancreatic trypsin inhibitor (Mr 6,000) binds to and inhibitstrypsin; a r antiproteinase (Mr 53,000) primarily inhibits elastase. Aninsufficiency of ai-antiproteinase, believed to be caused by exposure tocigarette smoke, leads to lung damage and the condition known asemphysema.Other examples of zymogen activation occur in hormones, connective tissue, and the blood-clotting system.
The hormone insulin is produced by cleavage of proinsulin, collagen is initially synthesized as asoluble precursor called procollagen, and blood clotting is mediated bya complicated cascade of zymogen activations.SummaryVirtually every biochemical reaction is catalyzedby enzymes. With the exception of a few catalyticRNAs, all known enzymes are proteins. Enzymesare extraordinarily effective catalysts, commonlyproducing reaction rate enhancements of 107 to1014.
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