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Formaldehyde damages many tissues, andblindness is a common result because the eyes are particularly sensitive. Ethanol competes effectively with methanol as a substrate foralcohol dehydrogenase. The therapy for methanol poisoning is intravenous infusion of ethanol, which slows the formation of formaldehydesufficiently so that most of the methanol can be excreted harmlessly inthe urine.Two other types of reversible inhibition, noncompetitive and uncompetitive, are often defined in terms of one-substrate enzymes but inpractice are only observed with enzymes having two or more substrates. A noncompetitive inhibitor is one that binds to a site distinct from that which binds the substrate (Fig.

8-15); inhibitor bindingdoes not block substrate binding (or vice versa). The enzyme is inactivated when inhibitor is bound, whether or not substrate is also present. The inhibitor effectively lowers the concentration of active enzymeand hence lowers the apparent V max (Vmax = k cat [EtJ). There is oftenChapter 8 EnzymesBOX 8-2221Kinetic Tests for Determining Inhibition MechanismsThe double-reciprocal plot (see Box 8-1) offers aneasy way of determining whether an enzyme inhibitor is competitive or noncompetitive. Two sets ofrate experiments are carried out, in both of whichthe enzyme concentration is held constant.

In thefirst set, [SJ is also held constant, permitting measurement of the effect of increasing inhibitor concentration [I] on the initial rate Vo (not shown). Inthe second set, [I] is held constant but [S] is varied.In the double-reciprocal plot VV0 is plotted versusMS].Figure 1 shows a set of double-reciprocal plotsobtained in the absence of the inhibitor and withtwo different concentrations of a competitive inhibitor. Increasing [I] results in the production of afamily of lines with a common intercept on the VVoaxis but with different slopes. Because the intercept on the 1/Vo axis is equal to 1/Vmax, we can seethat Vmax is unchanged by the presence of a competitive inhibitor.

That is, regardless of the concentration of a competitive inhibitor, there is alwayssome high substrate concentration that will displace the inhibitor from the enzyme's active site.In noncompetitive inhibition, similar plots ofthe rate data give the family of lines shown in Figure 2, having a common intercept on the 1/[S] axis.This indicates that Km for the substrate is not altered by a noncompetitive inhibitor, but Vmax decreases.[I][I][S] VmM,Figure 1 Competitive inhibition.little or no effect on Km. These characteristic effects of a noncompetitive inhibitor are further analyzed in Box 8-2. An uncompetitiveinhibitor (Fig. 8-15) also binds at a site distinct from the substrate.However, an uncompetitive inhibitor will bind only to the ES complex.(The noncompetitive inhibitor binds to either free enzyme or the EScomplex.)With these definitions in mind, consider a bisubstrate enzyme withseparate binding sites within the active site for two substrates, Si andS2, and suppose an inhibitor (I) binds to the site for S2.

If Si and S 2normally bind to the enzyme independently (in random order), I mayact as a competitive inhibitor of S2. However, since I binds at a sitedistinct from the site for Si, but will exclude S 2 and thereby block thereaction of Si, I may act as a noncompetitive inhibitor of Si. Alternatively, if Si normally binds to the enzyme before S 2 (ordered binding),then I may bind only to the ESi complex and act as an uncompetitiveinhibitor of Si. These are only a few of the scenarios that can be encountered with reversible inhibition of bisubstrate enzymes, and theeffects of these inhibitors can provide much information about reactionmechanisms.Figure 2 Noncompetitive inhibition.222Part II Structure and CatalysisFigure 8—16 Reaction of chymotrypsin withdiisopropylfluorophosphate (DIFP).

This reactionled to the discovery that Ser195 is the key activesite serine. DIFP also acts as a poison nerve gasbecause it irreversibly inactivates the enzyme acetylcholinesterase by a mechanism similar to thatshown here. Acetylcholinesterase cleaves the neurotransmitter acetylcholine, an essential step in normal functioning of the nervous system.OEnz—CH2—OH + F — P - O - C H(Ser1ocoCH3En/ - C H 2 —O—P—O-CHOCH,CDIFPIrreversible inhibitors are those that combine with or destroy afunctional group on the enzyme that is essential for its activity. Formation of a covalent link between an irreversible inhibitor and an enzymeis common.

Irreversible inhibitors are very useful in studying reactionmechanisms. Amino acids with key catalytic functions in the activesite can sometimes be identified by determining which amino acid iscovalently linked to an inhibitor after the enzyme is inactivated. Anexample is shown in Figure 8-16.A very special class of irreversible inhibitors are the suicide inhibitors. These compounds are relatively unreactive until they bind tothe active site of a specific enzyme. A suicide inhibitor is designed tocarry out the first few chemical steps of the normal enzyme reaction.Instead of being transformed into the normal product, however, theinhibitor is converted to a very reactive compound that combines irreversibly with the enzyme.

These are also called mechanism-basedinactivators, because they utilize the normal enzyme reaction mechanism to inactivate the enzyme. These inhibitors play a central role inthe modern approach to obtaining new pharmaceutical agents, a process called rational drug design. Because the inhibitor is designed to bespecific for a single enzyme and is unreactive until within that enzyme's active site, drugs based on this approach are often very effectiveand have few side effects (see Box 21-1).pH(a)Enzyme Activity Is Affected by pH10pH(b)Figure 8-17 pH-activity profiles of two enzymes.Such curves are constructed from measurementsof initial velocities when the reaction is carried outin buffers of different pH.

The pH optimum for theactivity of an enzyme generally reflects the cellularenvironment in which it is normally found, (a) Pepsin, which hydrolyzes certain peptide bonds of proteins during digestion in the stomach, has a pHoptimum of about 1.6. The pH of gastric juice is between 1 and 2. (b) Glucose-6-phosphatase of hepatocytes, with a pH optimum of about 7.8, is responsible for releasing glucose into the blood.

The normalpH of the cytosol of hepatocytes is about 7.2.Enzymes have an optimum pH or pH range in which their activity ismaximal (Fig. 8-17); at higher or lower pH their activity decreases.This is not surprising because some amino acid side chains act as weakacids and bases that perform critical functions in the enzyme activesite. The change in ionization state (titration) of groups in the activesite is a common reason for the activity change, but it is not the onlyone. The group being titrated might instead affect some critical aspectof the protein structure.

Removing a proton from a His residue outsidethe active site might, for example, eliminate an ionic interaction essential for stabilization of the active conformation of the enzyme. Lesscommon are cases in which the group being titrated is on the substrate.The pH range over which activity changes can provide a clue as towhat amino acid is involved (see Table 5-1). A change in enzyme activity near pH 7.0, for example, often reflects titration of a His residue.The effects of pH must be interpreted with some caution, however. Inthe closely packed environment of a protein, the pK of amino acid sidechains can change significantly.

For example, a nearby positive chargecan lower the pK of a Lys residue, and a nearby negative charge canincrease its pK. Such effects sometimes result in a pifthat is perturbedby 2 or more pH units from its normal value. One Lys residue in theenzyme acetoacetate decarboxylase has a pi^of 6.6 (10.5 is normal) dueto electrostatic effects of nearby positive charges.1316Examples of Enzymatic ReactionsThis chapter has focused on the general principles of catalysis and anintroduction to some of the kinetic parameters used to describe enzymeaction. Principles and kinetics are combined in Box 8-3, which describes some of the evidence that reinforces the notion that bindingenergy and transition-state complementarity are central to enzymaticcatalysis.

We now turn to several examples of specific enzyme reactionmechanisms.An understanding of the complete mechanism of action of a purified enzyme requires a knowledge of (1) the temporal sequence inwhich enzyme-bound reaction intermediates occur, (2) the structure ofeach intermediate and transition state, (3) the rates of interconversionbetween intermediates, (4) the structural relationship of the enzymewith each intermediate, and (5) the energetic contributions of all reacting and interacting groups with respect to intermediate complexes andtransition states.

There is probably no enzyme for which current understanding meets this standard exactly. Many decades of research,however, have produced mechanistic information about hundreds ofenzymes, and in some cases this information is highly detailed.A Chain22342His5758B ChainAsp102122136146149168182C Chain191201Reaction Mechanisms Illustrate PrinciplesMechanisms are presented for three enzymes: chymotrypsin, hexokinase, and tyrosyl-tRNA synthetase. These are chosen not necessarilybecause they are the best-understood enzymes or cover all possibleclasses of enzyme chemistry, but because they help to illustrate somegeneral principles outlined in this chapter.

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