Examples of Enzyme-Catalyzed Reactions

Chymotrypsin is an enzyme that catalyzes the hydrolysis of peptide bonds, with some specihcity for residues containing aromatic side chains. Chymotrypsin also cleaves peptide bonds at other sites, such as leucine, histidine, and glutamine, but with a lower frequency than at aromatic amino acid residues. It also catalyzes the hydrolysis of ester bonds. 

Although ester hydrolysis is not important to the physiological role of chy-motrypsin in the digestion of proteins, it is a convenient model system for investigating the enzyme s catalysis of hydrolysis reactions. The usual laboratory procedure is to use / -nitrophenyl esters as the substrate and to monitor the progress of the reaction by the appearance of a yellow color in the reaction mixture caused by the production of /(-nitrophenolate ion. 

Another enzyme-catalyzed reaction is the one catalyzed by the enzyme aspartate transcarbamoylase (ATGase). This reaction is the first step in a pathway leading to the formation of cytidine triphosphate (GTP) and uridine triphosphate (UTP), which are ultimately needed for the biosynthesis of RNA and DNA. In this reaction, carbamoyl phosphate reacts with aspartate to produce carbamoyl aspartate and phosphate ion. 

Garbamoyl phosphate + Aspartate Garbamoyl aspartate + HPO Reaction catalyzed by aspartate transcarbamoylase 

The rate of this reaction also depends on substrate concentration—in this case, the concentration of aspartate (the carbamoyl phosphate concentration is kept constant). Experimental results show that, once again, the rate of the reaction depends on substrate concentration at low and moderate concentrations, and, once again, a maximum rate is reached at high substrate concentrations. 

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Ligase reactions provide remarkable examples of enzyme-catalyzed reactions with 6 to 8 reactants. Since C = N -R at specified pH and C = Af"- "at specified pH and specified availability of oxygen atoms, this means there are 5 to 7 components. Three or four conservation equations are provided by elements, and so these enzyme mechanisms introduce 2 to 4 or 1 to 3 conservation equations in addition in coupling. In this respect enzyme-catalyzed reactions can be very different from chemical reactions (see Chapter 7). 

Examples of Enzyme-Catalyzed Reactions and Their Treatment... 

As we look at some examples of enzyme-catalyzed reactions, notice that the functional groups on the enzyme side chains are the same functional groups you are used to seeing in simple organic compounds, and the modes of catalysis used by enzymes are the same as the modes of catalysis used in organic reactions. The remarkable catalytic ability of enzymes stems in part from their ability to use several modes of catalysis in the same reaction. Factors other than those listed can contribute to the increased rate of enzyme-catalyzed reactions, but not all factors are employed by every enzyme. We will consider some of these factors when we discuss individual enzymes. Now let s look at the mechanisms for five enzyme-catalyzed reactions. 

Animals and plants cannot synthesize vitamin B12. In fact, only a few microorganisms can synthesize it. Humans must obtain all their vitamin B12 from their diet, particularly from meat. Because vitamin B12 is needed in only very small amounts, deficiencies caused by consumption of insufficient amounts of the vitamin are rare, but have been found in vegetarians who eat no animal products. Deficiencies are most commonly caused by an inability to absorb the vitamin in the intestine. The deficiency causes pernicious anemia. The following are examples of enzyme-catalyzed reactions that require coenzyme B12 ... 

The use of a lipase to carry out ester syndiesis is one of the earliest examples of enzyme-catalyzed reactions in organic media (1). A voluminous amount of papers has been published in the literature, iiKluding several reviews and books (2). In the polymer and biomaterials areas, lipases and esterases are... 

The following are examples of enzyme-catalyzed reactions that require coenzyme B12. 

Parameter setup for Example 9.1. Rate of enzyme-catalyzed reaction... 

Typically the reaction was carried out as follows to a mixture of lipase in the IL were added this racemic alcohol and vinyl acetate as the acyl donor. The resulting mixture was stirred at 35°C and the reaction course was monitored by GC analysis. After the reaction, ether was added to the reaction mixture to form a biphasic layer, and product acetate and unreacted alcohol were extracted with ether quantitatively. The enzyme remained in the IL phase as expected (Fig. 2). Two months later, Kim and co-workers reported similar results and Lozano and Ibora " reported other examples of lipase-catalyzed reaction in June. Further Park and Kazlauskas reported full details of lipase-catalyzed reaction in an IL solvent... 

For these reasons, in the experimental study of the kinetics of enzyme-catalyzed reactions, T, shear and PH are carefully controlled, the last by use of buffered solutions. In the development, examples, and problems to follow, we assume that both T and pH... 

Abstract This chapter introduces the basic principles used in applying isotope effects to studies of the kinetics and mechanisms of enzyme catalyzed reactions. Following the introduction of algebraic equations typically used for kinetic analysis of enzyme reactions and a brief discussion of aqueous solvent isotope effects (because enzyme reactions universally occur in aqueous solutions), practical examples illustrating methods and techniques for studying enzyme isotope effects are presented. Finally, computer modeling of enzyme catalysis is briefly discussed. 

Just as in the preceding examples, early indications of tunneling in enzyme-catalyzed reactions depended on the failure of experiments to conform to the traditional expectations for kinetic isotope effects (Chart 3). Table 1 describes experimental determinations of -secondary isotope effects for redox reactions of the cofactors NADH and NAD. The two hydrogenic positions at C4 of NADH are stereochemically distinct and can be labeled individually by synthetic use of enzyme-catalyzed reactions. In reactions where the deuterium label is not transferred (see below), an... 

An increasing number of enzyme-catalyzed reactions normally occurring in seconds to minutes have been successfully investigated in mixed solvents at subzero temperatures and have been resolved in time, step by step. Some examples are presented in the following sections. 

The kinetics of enzyme-catalyzed reactions (i. e the dependence of the reaction rate on the reaction conditions) is mainly determined by the properties of the catalyst, it is therefore more complex than the kinetics of an uncatalyzed reaction (see p.22). Here we discuss these issues using the example of a simple first-order reaction (see p.22)... 

Finally, yet another issue enters into the interpretation of nonlinear Arrhenius plots of enzyme-catalyzed reactions. As is seen in the examples above, one typically plots In y ax (or. In kcat) versus the reciprocal absolute temperature. This protocol is certainly valid for rapid equilibrium enzymes whose rate-determining step does not change throughout the temperature range studied (and, in addition, remains rapid equilibrium throughout this range). However, for steady-state enzymes, other factors can influence the interpretation of the nonlinear data. For example, for an ordered two-substrate, two-product reaction, kcat is equal to kskjl ks + k ) in which ks and k are the off-rate constants for the two products. If these two rate constants have a different temperature dependency (e.g., ks > ky at one temperature but not at another temperature), then a nonlinear Arrhenius plot may result. See Arrhenius Equation Owl Transition-State Theory van t Hoff Relationship... 

Except for very simple systems, initial rate experiments of enzyme-catalyzed reactions are typically run in which the initial velocity is measured at a number of substrate concentrations while keeping all of the other components of the reaction mixture constant. The set of experiments is run again a number of times (typically, at least five) in which the concentration of one of those other components of the reaction mixture has been changed. When the initial rate data is plotted in a linear format (for example, in a double-reciprocal plot, 1/v vx. 1/[S]), a series of lines are obtained, each associated with a different concentration of the other component (for example, another substrate in a multisubstrate reaction, one of the products, an inhibitor or other effector, etc.). The slopes of each of these lines are replotted as a function of the concentration of the other component (e.g., slope vx. [other substrate] in a multisubstrate reaction slope vx. 1/[inhibitor] in an inhibition study etc.). Similar replots may be made with the vertical intercepts of the primary plots. The new slopes, vertical intercepts, and horizontal intercepts of these replots can provide estimates of the kinetic parameters for the system under study. In addition, linearity (or lack of) is a good check on whether the experimental protocols have valid steady-state conditions. Nonlinearity in replot data can often indicate cooperative events, slow binding steps, multiple binding, etc. 

NADH. These experiments were pioneering with respect to contemporary enzymology, especially with regard to early recognition that coenzymes are held within enzyme active sites in stereochemically preferred ways. One typically utilizes NADH that contains a tritium or deuterium atom in the 4R or 45 position, and the success or failure of substrate deuteration/tritiation indicates the stereochemistry. Westheimer has tabulated the known examples of dehydrogenases that exhibit specificity for a particular face of NADH. Creighton and Murthy have reproduced this tabulation in their comprehensive review on the stereochemistry of enzyme-catalyzed reactions at carbon. 

A class of compounds in which a positively charged atom from group V or VI of the periodic table (c.g., N, O, S, P, As, Se) is bonded to a carbon atom having an unshared pair of electrons. Whereas there is only one canonical form for nitrogen ylides (R3N —CR2 ), because of pTT-dTT bonding, two canonical forms can be written for phosphorus Le., R3P=CR2 R3P —CR2 ) and sulfur ylides (R2S=CR2 R3S —CR2 ). A number of enzyme-catalyzed reactions have been reported to utilize ylide-based chemistry. For example, the ylide form of the... 

The use of MALDI-MS for the measurement of low molecular mass compounds is widely accepted now [61], but quantification remains problematic. The main problem is the inhomogeneous distribution of the analytes within the matrix [62]. This leads to different amounts of ions and therefore to different signal intensities at various locations of a sample spot. The simplest and most effective way to overcome this problem is the use of an appropriate internal standard [63]. The use of deuterated compounds with a high molecular similarity to the analyte as internal standards leads to a linear correlation between relative signal intensities and relative amount of the compound to be quantified (Fig. 4b) [64]. Using this approach it is possible to quantitate substrates and products of enzyme catalyzed reactions. Two examples were shown recently by Kang and coworkers [64, 65]. The first was a lipase catalyzed reaction which produces 2-methoxy-N-[(lR)-l-phenylethyl]-acetamide (MET) using rac-a-... 

Upon use of structurally modified variants as internal standards for the particular analytes, the relative quantificahon of oligonucleotides, peptides, and small proteins was demonstrated [44]. The potential of the ILM to allow quantitative analyses of peptides without the use of internal standards was presented recently [43]. Linear correlahons between peptide amount and signal intensities could be found upon applicahon of increased matrix-to-analyte ratios between 25,000 and 250,000 (mokmol). The dynamic range of linearity thus spanned one order of magnitude. Unfortunately, the importance of the M/A ratio prevents the use of this method in samples with unknown orders of concentration, for example, in a proteomics environment. On the other hand, the method is applicable for the screening of enzyme-catalyzed reactions because the starting concentrahons of the peptides are generally known in such assays. 

Phosphonates have been widely used as analogues of carboxylic acids. They have been particularly effective as analogues of tetrahedral transition states that occur in the course of enzyme-catalyzed reactions such as hydrolysis of the amide (peptide) bond. As such, they may be used as inhibitors of enzymes (e.g., 82, 83) or as haptens for producing antibodies that are catalytic (e.g., 84). A notable example is H203P— CH2—CH2—CH(—NH2)—COOH, which has effects that are likely to be due to its interference with glutamate as a neurotransmitter (85). 

The reactants of enzyme-catalyzed reactions are termed substrates and each enzyme is quite specific in character, acting on a particular substrate or substrates to produce a particular product or products. The names of enzymes usually indicate the substrate involved. For example, hydrogen peroxide oxidoreductase is an enzyme that uses hydrogen peroxide as its substrate to carry out the oxidation of organic substrates. Such formal names are often abbreviated such as in the contraction of hydrogen peroxide oxidoreductase to peroxidase. However, in the interest of avoiding... 

Structural studies of the oxy-Cope catalytic antibody system reinforce the idea that conformational dynamics of both protein and substrate are intimately intertwined with enzyme catalysis, and consideration of these dynamics is essential for complete understanding of biologically catalyzed reactions. Indeed, recent single molecule kinetic studies of enzyme-catalyzed reactions also suggest that different conformations of proteins are associated with different catalytic rates (Xie and Lu, 1999). In addition, a number of enzymes are known to undergo conformational changes on binding of substrate (Koshland, 1987) that lead to enhanced catalysis two examples are hexokinase (Anderson and Steitz, 1975 Dela-Fuente and Sols, 1970) and triosephosphate isomerase (Knowles, 1991). 

The activating cation presumably acts by helping the protein to maintain a productive conformation, and examples are provided by structural studies of enzymes that exhibit this property. Pauling suggested that complementarity between the stracture of the enzyme active site and the transition state of the reaction is responsible for the lowering of activation energies of enzyme-catalyzed reactions. Cation activation may aid this by ensuring that the active site has the correct... 

Comprehensive kinetic analysis defines the mechanistic basis for enzyme specificity and efficiency in ways that can be directly related to enzyme structure. In this article, the rationale will be described for design and interpretation of experiments to define the pathway of enzyme-catalyzed reactions using transient kinetic methods. These principles will be illustrated with three examples of biologically important reactions, none of which could have been solved with steady-state kinetics alone. This article is by no means a comprehensive survey of this extensive field, but rather, selected examples from the author s laboratory will be used to illustrate the methods to provide a flavor for what is possible. 

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