Enzyme kinetics mutations effect

Recently, a controversial debate has arisen about whether the optimization of enzyme catalysis may entail the evolutionary implementation of chemical strategies that increase the probability of tunneling and thereby accelerate reaction rates [7]. Kinetic isotope effect experiments have indicated that hydrogen tunneling plays an important role in many proton and hydride transfer reactions in enzymes [8, 9]. Enzyme catalysis of horse liver alcohol dehydrogenase may be understood by a model of vibrationally enhanced proton transfer tunneling [10]. Furthermore, the double proton transfer reaction in DNA base pairs has been studied in detail and even been hypothesized as a possible source of spontaneous mutation [11-13]. 

An interesting and potentially useful observation in the mutational work is that the substitution of leucine for Hisl43o decreased the activity to 0.2% of wild type. This significant activity allowed the deuterium kinetic isotope effect for the reaction of [ 1- H2] propane-1,2-diol to be measured. The value obtained turned out to be 2 for ° cat, about one-fifth to one-sixth of that for wild-type enzyme. Substitution of alanine gave about 1.5% activity and a deuterium kinetic isotope effect of 5-6. It appears that the hydrogen transfer is not solely rate limiting in these variants. 

Site-directed mutagenesis is an indispensable technique for determining the effect of substituting a specific amino acid writh another. For example, enzyme reaction rates can be measured for both wdld type and mutant enzymes, and changes in enzyme kinetics can be monitored to assess the possible catalytic role of a given residue. The complete absence of activity in a mutant enzyme indicates that the mutated residue is essential for catalysis. 

We can now relate the kinetic constants kCM, Ku, and kcJKM to specific portions of the enzyme reaction mechanism. From our discussions above we have seen that the term kCM relates to the reaction step of ES conversion to ES. Hence experimental perturbations (e.g., changes in solution conditions, changes in substrate identity, mutations of the enzyme, and the presence of a specific inhibitor) that exclusively affect kCM are exerting their effect on catalysis at the ES to ES transition step. The term KM relates mainly to the dissociation reaction of the encounter complex ES returning to E + S. Conversely, the reciprocal of Ku (1IKU) relates to the association step of E and S to form ES. Inhibitors and other perturbations that affect the... 

One of the important consequences of studying catalysis by mutant enzymes in comparison with wild-type enzymes is the possibility of identifying residues involved in catalysis that are not apparent from crystal structure determinations. This has been usefully applied (Fersht et al., 1988) to the tyrosine activation step in tyrosine tRNA synthetase (47) and (49). The residues Lys-82, Arg-86, Lys-230 and Lys-233 were replaced by alanine. Each mutation was studied in turn, and comparison with the wild-type enzyme revealed that each mutant was substantially less effective in catalysing formation of tyrosyl adenylate. Kinetic studies showed that these residues interact with the transition state for formation of tyrosyl adenylate and pyrophosphate from tyrosine and ATP and have relatively minor effects on the binding of tyrosine and tyrosyl adenylate. However, the crystal structures of the tyrosine-enzyme complex (Brick and Blow, 1987) and tyrosyl adenylate complex (Rubin and Blow, 1981) show that the residues Lys-82 and Arg-86 are on one side of the substrate-binding site and Lys-230 and Lys-233 are on the opposite side. It would be concluded from the crystal structures that not all four residues could be simultaneously involved in the catalytic process. Movement of one pair of residues close to the substrate moves the other pair of residues away. It is therefore concluded from the kinetic effects observed for the mutants that, in the wild-type enzyme, formation of the transition state for the reaction involves a conformational change to a structure which differs from the enzyme structure in the complex with tyrosine or tyrosine adenylate. The induced fit to the transition-state structure must allow interaction with all four residues simultaneously. 

We have used a series of biocatalysts produced by site-directed mutations at the active site of L-phenylalanine dehydrogenase (PheDH) of Bacillus sphaericus, which expand the substrate specificity range beyond that of the wild-type enzyme, to catalyse oxidoreduc-tions involving various non-natural L-amino acids. These may be produced by enantiose-lective enzyme-catalysed reductive amination of the corresponding 2-oxoacid. Since the reaction is reversible, these biocatalysts may also be used to effect a kinetic resolution of a D,L racemic mixture. ... 

To probe the effects of the mutations on specificity at the Pi position, we used peptide substrates, Pro-Thr-Glu-Phe-Phe(4-N02)-Arg-Leu (PTEFF(4-N02)RL, peptide A ), Pro-Thr-Glu-Lys-Phe(4-N02)-Arg-Leu (PTEKF(4-N02)RL, peptide B ), and Ac-Ala-Ala-Lys-Phe(4-N02)-Ala-Ala-amide (Ac-AAKF(4-N02)AA-NH2, peptide C ). Peptide A was cleaved Phe-Phe(4-N02) bond, which represents pepsin-like activity, and peptide B and peptide C were cleaved at the Lys-Phe(4-N02) bond, which is equivalent to trypsinogen activation. No other bond in these peptides was hydrolyzed by any of the enzymes. From kinetic determination of... 

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