The Enzyme s

Figure C1.5.17.(A) Enzymatic cycle of cholesterol oxidase, which catalyses tire oxidation of cholesterol by molecular oxygen. The enzyme s naturally fluorescent FAD active site is first reduced by a cholesterol substrate,...

The earliest examples of analytical methods based on chemical kinetics, which date from the late nineteenth century, took advantage of the catalytic activity of enzymes. Typically, the enzyme was added to a solution containing a suitable substrate, and the reaction between the two was monitored for a fixed time. The enzyme s activity was determined by measuring the amount of substrate that had reacted. Enzymes also were used in procedures for the quantitative analysis of hydrogen peroxide and carbohydrates. The application of catalytic reactions continued in the first half of the twentieth century, and developments included the use of nonenzymatic catalysts, noncatalytic reactions, and differences in reaction rates when analyzing samples with several analytes. 

Km for an enzymatic reaction are of significant interest in the study of cellular chemistry. From equation 13.19 we see that Vmax provides a means for determining the rate constant 2- For enzymes that follow the mechanism shown in reaction 13.15, 2 is equivalent to the enzyme s turnover number, kcat- The turnover number is the maximum number of substrate molecules converted to product by a single active site on the enzyme, per unit time. Thus, the turnover number provides a direct indication of the catalytic efficiency of an enzyme s active site. The Michaelis constant, Km, is significant because it provides an estimate of the substrate s intracellular concentration. 

Elucidating Mechanisms for the Inhibition of Enzyme Catalysis An inhibitor interacts with an enzyme in a manner that decreases the enzyme s catalytic efficiency. Examples of inhibitors include some drugs and poisons. Irreversible inhibitors covalently bind to the enzyme s active site, producing a permanent loss in catalytic efficiency even when the inhibitor s concentration is decreased. Reversible inhibitors form noncovalent complexes with the enzyme, thereby causing a temporary de-... 

Eor measurement of a substrate by a kinetic method, the substrate concentration should be rate-limiting and should not be much higher than the enzyme s K. On the other hand, when measuring enzyme activity, the enzyme concentration should be rate-limiting, and consequentiy high substrate concentrations are used (see Catalysis). 

A given enzyme may be assayed by its action on soluble substrates under chemical and physical conditions different from those encountered in a real-life wash. Such experiments indicate the enzyme s performance with respect to pH and temperature variations, or in conjunction with other soluble substances, etc. The analytical data thus obtained are not necessarily representative of the wash performance of the enzyme, and real wash trials are necessary to evaluate wash performance of detergent enzymes. 

The often fast binding step of the inhibitor I to the enzyme E, forming the enzyme inhibitor complex E-I, is followed by a rate-determining inactivation step to form a covalent bond. The evaluation of affinity labels is based on the fulfillment of the following criteria (/) irreversible, active site-directed inactivation of the enzyme upon the formation of a stable covalent linkage with the activated form of the inhibitor, (2) time- and concentration-dependent inactivation showing saturation kinetics, and (3) a binding stoichiometry of 1 1 of inhibitor to the enzyme s active site (34). 

Porcine liver esterase (PLE) gives excellent enantioselectivity with both dimethyl 3-methylglutarate [19013-37-7] (lb) and malonate (2b) diester. It is apparent from Table 1 that the enzyme s selectivity strongly depends on the size of the alkyl group in the 2-position. The hydrolysis of ethyl derivative (2c) gives the S-enantiomer with 75% ee whereas the hydrolysis of heptyl derivative (2d) results in the R-monoester with 90% ee. Chymotrypsin [9004-07-3] (CT) does not discriminate glutarates that have small substituents in the 3-position well. However, when hydroxyl is replaced by the much bulkier benzyl derivative (Ic), enantioselectivity improves significantly. 

The transformations described thus far were catalyzed by enzymes in their traditional hydrolytic mode. More recent developments in the area of enzymatic catalysis in nonaqueous media (11,16,33—35) have significantly broadened the repertoire of hydrolytic enzymes. The acyl—enzyme intermediate formed in the first step of the reaction via acylation of the enzyme s active site nucleophile can be deacylated in the absence of water by a number of... 

The simplest kinetic scheme that can account for enzyme-catalyzed reactions is Scheme XX, where E represents the enzyme, S is the substrate, P is a product, and ES is an enzyme-substrate complex. 

Kinetics is the branch of science concerned with the rates of chemical reactions. The study of enzyme kinetics addresses the biological roles of enzymatic catalysts and how they accomplish their remarkable feats. In enzyme kinetics, we seek to determine the maximum reaction velocity that the enzyme can attain and its binding affinities for substrates and inhibitors. Coupled with studies on the structure and chemistry of the enzyme, analysis of the enzymatic rate under different reaction conditions yields insights regarding the enzyme s mechanism of catalytic action. Such information is essential to an overall understanding of metabolism. 

Reactions conforming to this kinetic pattern are characterized by the fact that the product of the enzyme s reaction with A (called P in the following schemes) is released prior to reaction of the enzyme with the second substrate, B. As a result of this process, the enzyme, E, is converted to a modified form, E, which then reacts with B to give the second product, Q, and regenerate the unmodified enzyme form, E ... 

The working hypothesis is that, by some means, interaction of an allosteric enzyme with effectors alters the distribution of conformational possibilities or subunit interactions available to the enzyme. That is, the regulatory effects exerted on the enzyme s activity are achieved by conformational changes occurring in the protein when effector metabolites bind. 

Viewed in this way, the best definition of rate enhancement depends upon the relationship between enzyme and substrate concentrations and the enzyme s kinetic parameters. 

FIGURE 19.8 At high [ATP], phosphofruc-tokinase (PFK) behaves cooperatively, and the plot of enzyme activity versus [frnctose-6-phos-phate] is sigmoid. High [ATP] thus inhibits PFK, decreasing the enzyme s affinity for frnc-tose-6-phosphate. 

Bennett, M. J., 1994. The enzyme.s of mitochondrial fatty acid oxidation. 

When either the organic solvent or the ionic liquid is used as pure solvent, proper control over the water content, or rather the water activity, is of crucial importance, as a minimum amount is necessary to maintain the enzyme s activity. For ionic liquids, a reaction can be operated at constant water activity by use of the same methods as established for organic solvents [17]. [BMIM][PFg] or [BMIM][(CF3S02)2N], for example, may be used as pure solvents and in biphasic systems. Water-miscible ionic liquids, such as [BMIM][BF4] or [MMIM][MeS04], can be used in the second case. 

The action of an enzyme. (A) An enzyme with substrate molecules C and DE. Note that the specific shape of the enzyme s active site matches the shape of the substrate. (B) The enzyme with the substrate molecules bound. (C) The enzyme, unchanged from its original form with the product molecules (CD and E). 

Unlike many of the catalysts that chemists use in the laboratory, enzymes are usually specific in their action. Often, in tact, an enzyme will catalyze only a single reaction of a single compound, called the enzyme s substrate. For example, the enzyme amylase, found in the human digestive tract, catalyzes only the hydrolysis of starch to yield glucose cellulose and other polysaccharides are untouched by amylase. 

Consider reaction schemes for the production of L-phenylalanine by enzymatic methods. Now match each of the following substrates with the enzyme(s) responsible for L-phenylalanine formation. 

One form of biological poisoning mirrors the effect of lead on a catalytic converter. The activity of an enzyme is destroyed if an alien substrate attaches too strongly to the enzyme s active site, because then the site is blocked and made unavailable to the true substrate (Fig. 13.42). As a result, the chain of biochemical reactions in the cell stops, and the cell dies. The action of nerve gases is believed to stem from their ability to block the enzyme-controlled reactions that allow impulses to travel through nerves. Arsenic, that favorite of fictional poisoners, acts in a similar way. After ingestion as As(V) in the form of arsenate ions (As043 ), it is reduced to As(III), which binds to enzymes and inhibits their action. 

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