Pyridoxal phosphate (PLP)-dependent enzymes are unrivaled in the diversity of reactions that they catalyze. New structural data have paved the way for targeted mutagenesis and mechanistic studies and have provided a framework for interpretation of those results. Together, these complementary approaches yield new insight into function, particularly in understanding the origins of substrate and reaction type specificity. The combination of new sequences and structures enables better reconstruction of their evolutionary heritage and illuminates unrecognized similarities within this diverse group of enzymes. The important metabolic roles of many PLP-dependent enzymes drive efforts to design specific inhibitors, which are now guided by the availability of comprehensive structural and functional databases. Better understanding of the function of this important group of enzymes is crucial not only for inhibitor design, but also for the design of improved protein-based catalysts.

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The possibility that the rates of acylation of chymotrypsin by certain highly reactive substrates approach the diffusion-controlled limits was investigated by measuring the values of kcat/Km for three substrates as a function of increasing viscosity with sucrose and ficoll as the viscosogenic reagents. The values of Kcat/Km (pH 8.0, 25 degrees C) representing the acylation rate constants are the following: N-(methoxycarbonyl)-L-tryptophan p-nitrophenyl ester, 3.5 x 10(7) M-1 s-1; N-acetyl-L-tryptophan methyl ester, 8 x 10(5) M-1 s-1; N-acetyl-L-tryptophan p-nitroanilide, 300 M-1 s-1. The rate constants decrease significantly with increasing viscosity for the first compound, decrease slightly for the second, and are insensitive to this perturbation for the third. The p-nitroanilide results taken together with the observation that the high concentrations of sucrose or ficoll used produce insignificant changes in kcat for the ester substrates argue against a general nonspecific perturbation in the enzyme structure effected by these reagents. The values of the association rate constants calculated from these results are 9 x 10(7) and 1 x 10(7) M-1 s-1 for the p-nitrophenyl and methyl esters, respectively. The values of kcat/Km divided by the association rate constants show that the rates of acylation by the p-nitrophenyl ester occur at ca. 40% and by the methyl ester at ca. 10% of the diffusion limits. Possibilities involving reorientation of a nonproductively bound substrate within the ES complex or desolvation of part of the active site of the enzyme are considered to account for the lower association rate constant for the methyl as compared to the p-nitrophenyl ester.

A true Brønsted analysis of proton transfer in an enzyme mechanism is made possible by the chemical rescue of an inactive mutant of aspartate aminotransferase, where the endogenous general base, Lys258, is replaced with Ala by site-directed mutagenesis. Catalytic activity is restored to this inactive mutant by exogenous amines. The eleven amines studied generate a Brønsted correlation with beta of 0.4 for the transamination of cysteine sulfinate, when steric effects are included in the regression analysis. Localized mutagenesis thus allows the classical Brønsted analysis of transition-state structure to be applied to enzyme-catalyzed reactions.

Dicer, a member of the ribonuclease III family of enzymes, processes double-stranded RNA substrates into approximately 21- to 27-nt products that trigger sequence-directed gene silencing by RNA interference. Although the mechanism of RNA recognition and length-specific cleavage by Dicer has been established, the way in which dicing activity is regulated is unclear. Here, we show that the N-terminal domain of human Dicer, which is homologous to DExD/H-box helicases, substantially attenuates the rate of substrate cleavage. Deletion or mutation of this domain activates human Dicer in both single- and multiple-turnover assays. The catalytic efficiency (k(cat)/K(m)) of the deletion construct is increased by 65-fold over that exhibited by the intact enzyme. Kinetic analysis shows that this activation is almost entirely due to an enhancement in k(cat). Modest stimulation of catalysis by the full-length Dicer enzyme was observed in the presence of the TAR-RNA binding protein, which physically interacts with the DExD/H-box domain. These results suggest that the DExD/H-box domain likely disrupts the functionality of the Dicer active site until a structural rearrangement occurs, perhaps upon assembly with its molecular partners.

The roles of the catalytic active-site residues aspartic acid-52 and glutamic acid-35 of chicken lysozyme (EC 3.2.1.17) have been investigated by separate in vitro mutagenesis of each residue to its corresponding amide (denoted as D52N and E35Q, respectively). The mutant enzyme D52N exhibits =5% of the wild-type lytic activity against Micrococcus luteus cell walls, while there is no measurable activity associated with E35Q (0.1% ± 0.1%). The measured dissociation constants for the chitotriose-enzyme complexes are 4.1 ,LM (D52N) and 13.4 pzM (E35Q) vs. 8.6 ,uM for wild type, indicating that the alterations in catalytic properties may be due in part to binding effects as well as to direct catalytic participation of these residues. The mutant lysozymes have been expressed in and secreted from yeast and obtained at a level of =a5 mg per liter of culture by high-salt elution from the cell walls.Chicken egg white lysozyme (CEWL; EC 3.2.1.17) has a distinguished history in the field of mechanistic enzymology. It was the first enzyme for which an atomic resolution x-ray structure was published (1), and the essence of the presently accepted catalytic mechanism was proposed almost exclusively from the structural information (2). The enzyme has additionally served as an important paradigm for studies in (i) the physical biochemistry of polydentate liganding, whereby the individual contributions to the several binding subsites have been separately evaluated (3); (ii) molecular evolution investigations (4, 5); and (iii) immunochemical studies (6). The enzyme is thus an attractive candidate for modification by site-directed mutagenesis, because the availability of specifically mutated constructs will have the potential to contribute significantly to all of the above investigations, as well as to studies on protein folding and stability. We report here the relatively high-efficiency expression of Strains. Plasmids were propagated in HB101 grown in LB medium (7). M13mpl8 subclones were grown in JM103 and in RZ1032 for mutagenesis (8). Saccharomyces cerevisiae strain GRF180 is leu2-3,-J12, ura3-52, his4-580, can (cir") and was derived from GRF18 by curing this strain of its endogenous 2-,um circle (9, 10). Escherichia coli and S. cerevisiae transformations were carried out as described (11,12).Plasmids. Plasmid pAB24 is a yeast-E. coli shuttle vector derived from pBR322 and pJDB219 (13). It contains the complete 2-gm circle and the LEU2-D and URA3 genes for selection and replication in yeast and pBR322 sequences and sequences for selection and replication in E. coli (14). Plasmid pAGAP1 is a derivative of pPGAP1 (15) that has the GAPDH-491 promoter replaced with a hybrid ADH2-GAPDH promoter fusion (14). Plasmid pLS1023 containing a CEWL cDNA clone (16) was the generous gift of G. Schutz (University of Cologne). DNA sequencing revealed two discrepancies between pLS1023 and the published sequence, which were repaired by site-directed mutagenesis.Site-Directed Mutagenesis of the Active Site. Two 21-base synthetic primers ...

Alanine scanning mutagenesis of the HyHEL-10 paratope of the HyHEL-100HEWL complex demonstrates that the energetically important side chains~hot spots! of both partners are in contact. A plot of DDG HyHEL-10_mutant vs. ⌬⌬G HEWL_mutant for the five of six interacting side-chain hydrogen bonds is linear~Slope ϭ 1!. Only 3 of the 13 residues in the HEWL epitope contribute Ͼ4 kcal0mol to the free energy of formation of the complex when replaced by alanine, but 6 of the 12 HyHEL-10 paratope amino acids do. Double mutant cycle analysis of the single crystallographically identified salt bridge, D32 H 0K97, shows that there is a significant energetic penalty when either partner is replaced with a neutral side-chain amino acid, but the D32 H N0K97M complex is as stable as the WT. The role of the disproportionately high number of Tyr residues in the CDR was evaluated by comparing the DDG values of the Tyr r Phe vs. the corresponding Tyr r Ala mutations. The nonpolar contacts in the light chain contribute only about one-half of the total DDG observed for the Tyr r Ala mutation, while they are significantly more important in the heavy chain. Replacement of the N31 L 0K96 hydrogen bond with a salt bridge, N31D L 0K96, destabilizes the complex by 1.4 kcal0mol. The free energy of interaction, DDG int , obtained from double mutant cycle analysis showed that DDG int for any complex for which the HEWL residue probed is a major immunodeterminant is very close to the loss of free energy observed for the HyHEL-10 single mutant. Error propagation analysis of double mutant cycles shows that data of atypically high precision are required to use this method meaningfully, except where large DDG values are analyzed.

The replacement of Lys258 by alanine (K258A) in aspartate aminotransferase reduces the rate constant for the central, 1,3-prototropic shift by 10(6)-10(8)-fold, confirming the role of Lys258 as the general-base catalyst for this step. The rate constant for the 1,3-prototropic shift interconverting K258A aldimine and ketimine intermediates is pH-independent like that of the wild-type enzyme (WT-AATase). K258A binds amino acid substrates in external aldimine intermediates 10(5)-fold more tightly than does WT-AATase. The excess amino acid binding energy observed in the mutant is sacrificed by the WT-AATase in order to increase the value of kcat. The net result is that the kcat/KM values for amino acid substrates are reduced only 3-100-fold by the mutation. This provides a clear example of the Circe effect propounded by Jencks [Jencks, W. P. (1975) Adv. Enzymol. Rel. Areas Mol. Biol. 43, 219]. Part of the increase in kcat due to the inclusion of Lys258 is accomplished by a 10(4)-10(5)-fold acceleration of external aldimine formation and hydrolysis. This step is partially rate-determining for K258A, but not for WT-AATase. A significant consequence of the utilization of amino acid binding energy for catalysis is the raising of the dissociation constants for these substrates to levels near the physiological concentrations of amino acids. The major product of the reaction of K258A with oxalacetate is pyruvate due to decarboxylation of the beta-imine formed in the ketimine intermediate.