Kinetics thermolysin

T4 lysozyme 33,497 helix stability of 528, 529 hydrophobic core stability of 533, 544 Tanford j8 value 544, 555, 578, 582-Temperature jump 137, 138, 541 protein folding 593 Terminal transferase 408,410 Ternary complex 120 Tertiary structure 22 Theorell-Chance mechanism 120 Thermodynamic cycles 125-131 acid denaturation 516,517 alchemical steps 129 double mutant cycles 129-131, 594 mutant cycles 129 specificity 381, 383 Thermolysin 22, 30,483-486 Thiamine pyrophosphate 62, 83 - 84 Thionesters 478 Thiol proteases 473,482 TNfn3 domain O-value analysis 594 folding kinetics 552 Torsion angle 16-18 Tbs-L-phenylalanine chloromethyl ketone (TPCK) 278, 475 Transaldolase 79 Tyransducin-o 315-317 Transit time 123-125 Transition state 47-49 definition 55... 

An important difference between thermolysin and carboxypeptidase leads to the major uncertainty in the mechanism of carboxypeptidase. This difference is that the catalytic carboxylate of carboxypeptidase is far more sterically accessible. The crucial question is whether or not the carboxypeptidase-catalyzed hydrolysis of peptides proceeds via general-base catalysis, as in equation 16.26, or via nucleophilic catalysis, as in 16.27. Early kinetic work concentrated on establishing the participation of the various groups in catalysis. 

Thermolysin belongs to a class of proteases (called neutral proteases) which are distinct from the serine proteases, sulfhydryl proteases, metal-loexopeptidases, and acid proteases. Neutral proteases A and B from Bacillus subtilis resemble thermolysin in molecular weight, substrate specificity, amino acid content, and metal ion dependence. Since physiological substrates are most likely proteins, it is difficult to design simple experiments that can be interpreted in terms of substrate specificity and relative velocities. Therefore, studies of substrate specificity and other kinetic parameters must be carried out on di- and tripeptides so that details of the mechanism of catalysis can be obtained and interpreted simply. 

The preceding study is directly applicable to understanding the active site of thermolysin, since a recent kinetic study (6) comparing neutral protease from B. subtilis with thermolysin showed that the two enzymes have identical pH rate profiles that peak near pH 7. 

K. Oyama, K. Kihara, and Y. Nonaka, On the mechanism of the action of thermolysin kinetic study of the thermolysin-catalyzed condensation reaction of N-benzyloxy-carbonyl-t-aspartic add with L-phenyl-alanine methyl ester, J. Chem. Soc. Perkin Trans. 2 1981a, 356-360. 

The kinetics of the binding of inhibitors to thermolysin appear to be controlled by the displacement of a water molecule from the active site (Holden et al., 1987). 

Trp, Leu, Met), and it was selected for making the Tyr-Gly, Phe-Leu, and Phe-Met peptide bonds. Papain was selected for Gly-Gly and Gly-Phe peptide bonds. Bromelain is very similar to papain as far as its specificity. All these proteases are serine or cysteine type, and the peptide bond formation can be done under kinetically controlled conditions. Thermolysin is an asparyl protease, and it was selected mainly for Phe-Leu and Gly-Phe bonds. MeCN, EtOAc, and methyl caproate were used as solvents with a controlled amount of buffer or at fixed water amount. 

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. 

The enzyme has also been used in the production of several natural amino acids such as L-serine from glycine and formaldehyde and L-tryptophan from glycine, formaldehyde, and indole [77-79], In addition, SHMT has also been used for the production of a precursor, 20, to the artificial sweetener aspartame (21) through a non-phenylalanine-requiring route (Scheme 14) [80-83]. Glycine methyl ester (22) is condensed with benzaldehyde under kinetically controlled conditions to form L-enY/ ra-p-phenylserine (23). This is then coupled enzymatically using thermolysin with Z-aspartic acid (24) to form A -carbobcnzyloxy-L-a-aspartyl-L-eryt/zro-p-phenylserine (20). and affords aspartame upon catalytic hydrogenation. 

Detailed information on the mechanism of biochemical reactions may be of crucial importance in designing new molecules having a pharmacological activity. For example, the detailed mechanism of protein hydrolysis by thermolysin has been studied at the QM/MM semiempirical level [25]. The various steps of the reaction and their transition states have been characterized. Fig. 3 (see color plate) shows the structure of the transition state of the rate-determining step. The important consequence of this approach is the fact that it is possible to evaluate the influence of the whole macro-molecular surroundings on the energetics of the process. It then becomes possible, for instance, to predict the influence of a mutation on the reaction kinetics. 

It is primarily for the above reasons that, in the view of this author, it is not yet possible to rmequivocably define the mechanistic role played by zinc ion for any zinc-enzyme. Nevertheless, with the exception of thermolysin, it is possible to arrive at reasonable mechanistic hypotheses for the various zinc enzymes considered in this review through the examination of data derived from both kinetic studies and from studies at equilibrium through judicious application of the anthropomorphic approach to the description of reaction mechanisms (50). 

Thermolysin is a potent endopeptidase isolated from the organism Bacillus thermoproteolyticus. In contrast to the other zinc metalloenzymes discussed in this review, thermolysin has not yet been subjected to rigorous chemical and kinetic investigation. Therefore, although the complete amino acid sequence is known [184), and although Matthews and co-workers [43 6) have carried the X-ray structural analysis to 2.3 A resolution, it is neither possible to discuss the thermolysin catalytic mechanism nor to propose a role for zinc ion in thermolysin catalysis. Therefore, the following discussion is restricted to a brief review of the current status of the physical and chemical properties of thermolysin, and to a... 

Using the reverse of the hydrolytic reaction, proteases are capable of catalyzing the formation of peptide bonds. Thermolysin is a powerful enzyme in catalyzing the synthesis of various useful oligopeptides, either in a free form or in an immobilized form in organic solvent media [58]. Recently, the systematic study about the stability of immobilized thermolysin has been reported [59]. In this study, the authors described the mechanism and kinetics for the inactivation of immobilized thermolysin with respect to the effects of organic solvents, kinds of support, water content, and so on. The strategy and results of their approach are discussed next. 

Many related so-called thermolysin-like proteinases (TLPs) from various Grampositive strains have been described [47], including neutral proteases from Bacillus subtilis, and some of these variants are applied in peptide synthesis. Several metal-loenzymes acting as carboxy- or aminopeptidase have also been characterized, but these variants have not been extensively used in peptide synthesis. A bovine carboxy-peptidase A [39] and orange carboxypeptidase C [68] have been applied for dipeptide synthesis in water-organic solvent mixtures, both under thermodynamic and xmder kinetic control. 

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