Substrate Binding and Catalysis

Active Site Catalytic Residues 1. Nucleophilic Catalysis 

In several cases, site-directed mutagenesis has helped to identify residues directly involved in acid-base catalysis. Deletion of residues which participate in acid-base catalysis leads to a marked reduction in catalytic activity at neutral pH and an alteration in the pH dependence for catalysis similar to results described 

The importance of optimal distance for proton transfer has been emphasized by work on triose-phosphate isomerase. An essential base, Glu-16S, has been replaced by Asp, effectively increasing the bond distance for proton transfer by 1 A (50). The rates of the enzyme-catalyzed enolization steps are reduced 1000-fold (50) relative to wild type. Although the mutant is impaired, its activity is still substantial considering that the wild-type enzyme accelerates the reaction 10 -fold relative to acetate ion in solution. Attempts to select for second-site revertants which restore catalytic activity have met with only modest success (51, 52), but they begin to address the important questions pertaining to the evolution of the optimal geometry of the constellation of amino acids around the active site. 

Effects of electrostatic interactions on substrate binding have been evaluated by amino acid substitution at two residues in the PI binding cleft of subtilisin, Glu-156 and Gly-166. The two residues are involved in two modes of substrate binding dependent on the properties of the PI substrate, thus contributing to the broad specificity of subtilisin. Measurements of ratios showed the antici- 

The importance of salt bridges at the NADPH-binding site of dihydrofolate reductase has been examined by a thorough analysis of two mutations, His-45 

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Molecular characteristics of luciferase. A molecule of the luciferase of G. polyedra comprises three homologous domains (Li et al., 1997 Li and Hastings, 1998). The full-length luciferase (135 kDa) and each of the individual domains are most active at pH 6.3, and they show very little activity at pH 8.0. Morishita et al. (2002) prepared a recombinant Pyrocystis lunula luciferase consisting of mainly the third domain. This recombinant enzyme catalyzed the light emission of luciferin (luminescence A.max 474 nm) and the enzyme was active at pH 8.0. The recombinant enzyme of the third domain of G. polyedra luciferase was crystallized and its X-ray structure was determined (Schultz et al., 2005). A -barrel pocket putatively for substrate binding and catalysis was identified in the structure, and... 

Li S, LP Wackett (1993) Reductive dehalogenation by cytochrome P450(,j jy[ substrate binding and catalysis. Biochemistry 32 9355-9361. 

Aleshin and coworkers (49) have reported the X-ray crystal structure at 2.2-A resolution of a G2-type variant produced by Aspergillus awamori. Meanwhile, an attempt was made to determine the amino acid residues that participate in the substrate binding and catalysis provided by G2 of A. niger (52). The results of the chemical approach indicated that the Asp-176, Glu-179, and Glu-180 form an acidic cluster crucial to the functioning of the enzyme. This conclusion was then tested by site-specific mutagenesis of these amino acid residues, which were replaced, one at a time, with Asn, Gin, and Gin, respectively (53). The substitution at Glu-179 provided an inactive protein. The other two substitutions affected the kinetic parameters but were not of crucial importance to the maintenance of activity. The crystal structure (49) supports the conclusion that Glu-179 functions as the catalytic acid but Asp-17 6 does not appear to be a good candidate for provision of catalytic base. Thus, there still exists considerable uncertainty as to how the disaccharide is accepted into the combining site for hydrolysis. Nevertheless, the kind of scheme presented by Svensson and coworkers (52) almost surely prevails. 

The plant soluble STs have around 25 to 30% amino acid identity with mammalian soluble STs, and are of a similar size. Comparisons between F. chloraefolia F3ST and F4 ST, combined with mutational analysis and data from the crystal structure of mouse estrogen ST, have defined amino acid residues important for PAPS binding, substrate binding and catalysis, and the mechanism of sulfonate transfer. " Sequence relatedness has been used to divide the STs into families and subfamilies in a similar manner as for P450s. ... 

Fig. 8.17. Mechanism of hydrolysis of phosphotyrosine residues by tyrosine phosphatases. Cleavage of phosphate from phosphotyrosine residues takes place by an in-line attack of a nucleophilic cysteine thiolate of the tyrosine phosphatase at the phosphate of the phosphotyrosine residue. The negative charge on the thiolate is stabilized by the positive charge of a conserved Arg residue. In the course of the reaction, an enzyme-Cys-phosphate intermediate is formed, which is hydrolytically cleaved to phosphate and enzyme-Cys-SH. The figure shows selected interactions. Other interactions in the active center involved in substrate binding and catalysis are not shown. According to Tainer and Russel, (1994). R substrate protein.

Catalytic Site. The catalytic site of COMT is a rather simple environment formed by the metal ion and by the amino acids important for substrate binding and catalysis of the methylation reaction. 

FIGURE 16-10 Iron-sulfur center in aconitase. The iron-sulfur center is in red, the citrate molecule in blue. Three Cys residues of the enzyme bind three iron atoms the fourth iron is bound to one of the carboxyl groups of citrate and also interacts noncovalently with a hydroxyl group of citrate (dashed bond). A basic residue ( B) on the enzyme helps to position the citrate in the active site. The iron-sulfur center acts in both substrate binding and catalysis. The general properties of iron-sulfur proteins are discussed in Chapter 19 (see Fig. 19-5). 

The first crystal structure of a bacterial serine protease to be solved—subtilisin, from Bacillus amyloliquefaciens—revealed an enzyme of apparently totally different construction from the mammalian serine proteases (Figure 1.17). This was not unexpected, since there is no sequence homology between them. But closer examination shows that they are functionally identical in terms of substrate binding and catalysis. Subtilisin has the same catalytic triad, the same system of hydrogen bonds for binding the carbonyl oxygen and the acetamido NH of the substrate, and the same series of subsites for binding the acyl portion of... 

This enzyme has been studied extensively by x-ray, kinetic, NMR, optical, circular dichroic, and fluorescence techniques. Thus, many approaches have been used to explore the role of the metal ions in catalysis and of other protein residues in substrate binding and catalysis. The review of this enzyme will serve to point out the information to be gained from using multiple biophysical approaches in understanding metalloenzyme catalysis. 

Kemp elimination was used as a probe of catalytic efficiency in antibodies, in non-specific catalysis by other proteins, and in catalysis by enzymes. Several simple reactions were found to be catalyzed by the serum albumins with Michaelis-Menten kinetics and could be shown to involve substrate binding and catalysis by local functional groups (Kirby, 2000). Known binding sites on the protein surface were found to be involved. In fact, formal general base catalysis seems to contribute only modestly to the efficiency of both the antibody and the non-specific albumin system, whereas antibody catalysis seems to be boosted by a non-specific medium effect. 

Amino Acids Involved in Substrate Binding and Catalysis... 

Smith, A. T., and Veitch, N. C., 1998, Substrate binding and catalysis in heme peroxidases, Curr. 

Nakajima K, Kato H, Oda J, Yamada Y, Hashimoto T. Site directed mutagenesis of putative substrate binding residues reveals a mechanism controlling different substrate specificities of two tropinone reductases. J. Biol. Chem. 1999 274 16563-16568. Yamashita A, Kato H, Wakatsuki S, Tomizaki T, Nakatsu T, Nakajima K, Hashimoto T, Yamada Y, Oda J. Structure of tropinone reductase-II with NADP + and pseudotropine at 1.9A resolution implication for stereospecific substrate binding and catalysis. Biochemistry 1999 38 7630-7637. 

The importance of the changes in quaternary structure in determining the sigmoidal curve is illustrated nicely by studies of the isolated catalytic trimer, freed by p-hydroxymercuribenzoate treatment. The catalytic subunit shows Michaelis-Menten kinetics with kinetic parameters that are indistinguishable from those deduced for the R state. Thus, the term tense is apt in the T state, the regulatory dimers hold the two catalytic trimers sufficiently close to one another that key loops on their surfaces collide and interfere with conformational adjustments necessary for high-affinity substrate binding and catalysis. 

The sequences of citrate synthases from the eukaryotes pig heart and kidney, Arabidopsis thaliana and Saccharomyces cerevisiae, and from the eubacteria Escherichia coli, Rickettsia prowazekii, Acinetobacter anitratum, Acetobacter aceti and Pseudomonas aeruginosa have been determined (see the literature [85,88] for references). In addition, a high-resolution X-ray crystallographic structure is available for the pig heart enzyme [89,90]. This has allowed the identification of 12 residues which are critical for substrate binding and catalysis multiple sequence alignments [91] have indicated that the majority of these 12 active site residues are conserved between all eukaryotic and eubacterial citrate synthases. 

The carboxyl-terminal extension of class II enzymes forms a hemicir-cular bannister around the calcium-binding loop. It is secured proximally (Cys-126 = Cys-27) and distally (Cys-134 = Cys-50) by disulfide bridges. The 7- or 8-residue loop is rich in prolines and charged residues. This substructure is remote from the residues implicated in interfacial ad-sorpdon, substrate binding, and catalysis and has no defined catalytic or pharmacological role. 

The molecular details of the action of metalloenzymes have begun to be elucidated in the past few years (42). Crystal structures for bovine carboxypeptidase A (43), thermolysin (44), and horse liver alcohol dehydrogenase (45) are now available, and chemical and kinetic studies have defined the role of zinc in substrate binding and catalysis. In fact, many of the significant features elucidating the mode of action of enzymes in general have been defined at the hands of zinc metalloenzymes. 

Guengerich FP, Hanna IH, Martin MV, Gillam EM. Role of glutamic acid 216 in cytochrome P450 2D6 substrate binding and catalysis. Biochemistry 2003 42 1245-53. 

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