Thermolysin mechanism

Similar reaction mechanisms, involving general base and metal ion catalysis, in conjunction with an OH nucleophilic attack, have been proposed for thermolysin (Ref. 12) and carboxypeptidase A (Refs. 12 and 13). Both these enzymes use Zn2+ as their catalytic metal and they also have additional positively charged active site residues (His 231 in thermolysin and... 

Evidence against the covalent mechanism has been summarized by Mock, who has also proposed alternative general-base-catalyzed mechanisms for ther-molysin and carboxypeptidase A.143 He suggests that His-231 is the general base for thermolysin and the carboxy-terminal carboxylate for carboxypeptidase A. The one common feature of all the proposed mechanisms is the Zn2+ functioning as a Lewis acid to polarize the substrate. 

Figure 12 Catalytic mechanism of thermolysin and stromelysin-1. (A) The mechanism of thermolysin [54], (B) The mechanism of stromleysin-1 [10]. Equivalent residues to Tyr-157 and His-231 are not observed for stromelysin-1. The proposed mechanism for collagenase-1 [S3] is similar to stromelysin-1, but also involves Asn-180 (equivalent to Asn-162 in stromelysin-1). This residue cannot participate in stromelysin-1 due to an additional residue between Ala-165 and Asn-162. (Adapted from Ref. 10.)...

The collapse of the proteolytic tetrahedral intermediate of the promoted-water pathway requires a proton donor in order to facilitate the departure of the leaving amino group. Rees and Lipscomb (1982) considered Glu-270, but favored Tyr-248 for this role, but Monzingo and Matthews (1984) fully elaborated on a role for Glu-270 of carboxypeptidase A and Glu-143 of thermolysin as intermediate proton donors. This proposal for carboxypeptidase A is corroborated by the near-normal activity observed for the Tyr-248- Phe mutant of rat carboxypeptidase A (Garden et al, 1985 Hilvert et al, 1986) and is reflected in the mechanistic scheme of Fig. 31 (Christianson and Lipscomb, 1989). Mock (1975) considered Glu-270 a proton donor in the carboxypeptidase A mechanism, but his mechanism does not favor a Glu-270/zinc-promoted water molecule as the hydrolytic nucleophile. Schepartz and Breslow (1987) observed that Glu-270 may mediate an additional proton transfer in the generation of the Pi product carboxylate. 

FIGURE 17. Favored reaction mechanism of thermolysin. Adapted with permission from References 9 and 140. Copyright (2003) and (1998) ACS... 

An 80- to 90-residue N-terminal propeptide domain contains a cysteine whose -S group binds to the active site zinc, screening it from potential substrates. The central catalytic domain is followed by a hinge region and a C-terminal domain that resembles the serum iron binding and transporting hemopexin.427/436 The mechanism of action is probably similar to that of thermolysin.430... 

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. 

Overall the mechanism of the thermolysin reaction including the role of the Zn2 + in catalysis is given in Fig. 5. This proposed mechanism is based on inhibitor data and x-ray structure determination. 

Fig. 5. Proposed mechanism of action of thermolysin showing the catalytically important residues.

Fig. 8 Mechanism of thermolysin and carboxypeptidase based on X-ray structures of enzymes with bound phosphonate inhibitors.34-37...

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. 

Thus, many metal ions catalyze the hydrolysis of esters [7,8], amides [9], and nitriles [10] via electrophilic activation of the C=0 or C=N group. This type of catalysis is characteristic of coordination complexes and is very common in metalloenzyme-mediated processes. Zinc(II), for example, is a key structural component of more than 300 enzymes, in which its primary function is to act as a Lewis acid (see Chapter 4). The mechanism of action of zinc proteases, e.g., thermolysin, involves electrophilic activation of an amide carbonyl group by coordination to zinc(II) in the active site (Figure 4). 

Alkaline phosphatases form a well-known class of proteins that perform quite interesting and complicated reactions. As previously reported, Zn enzymes, like carboxypeptidases, thermolysin, and carbonic anhydrases, consist of only one Zn atom per active center. Most of the alkaline phosphatases consist of two 96-kDa subunits, each containing two Zn and one Mg ion. The alkaline phosphatase from E. coli has been crystallized and described in full detail [4], and a mechanism has been proposed. Several enzymes in this category have been mentioned in recent years, some of them also containing different metal ions, such as iron and zinc, as in the purple acid phosphatase [5], It is likely that the detailed structure and mechanism of many more examples of enzymes that remove or add phosphate groups to proteins will become available in the next decade. 

The X-ray structures of enzyme and enzyme-inhibitor complexes permit the anhydride intermediate only in the case of carboxypeptidase, not in the case of thermolysin, since in this enzyme the catalytic Glu-143 is too far away from the substrate carbonyl (Lipscomb, 1983). The proposal that carboxypeptidase works via an anhydride intermediate thus requires the supposition that two very similar enzymes work by different mechanisms. 

Finally, enzymes that bind metal cofactors such as Zn + and Mg + can use their properties as Lewis acids, for example, electron pair acceptors. An example is the enzyme thermolysin, whose mechanism is illustrated in Fig. 9. In this enzyme, glutamate-143 acts as an active site base to deprotonate water for attack on the amide carbonyl, which is at the same time polarized by coordination by an active site Zn + ion (6). The protonated glutamic acid then probably acts as an acidic group for the protonation of the departing amine. 

The mechaiusm by which metalloproteinases execute catalysis has been of interest for many years. Most studies focused on carboxypeptidase A and thermolysin-like proteases for which extensive stmctural, chemical, and biochemical data are available. The first peptide hydrolysis mechanisms to be proposed... 

Standard mechanism inhibitors are classified strictly as inhibitors of serine proteases. There have been reports of inhibitors of other classes of proteases that have similar mechanisms to those of standard mechanism inhibitors, though. Initial studies on the streptomyces metalloprotease inhibitor (SMPl) suggest that it inhibits the metalloprotease thermolysin through a substrate-like binding mechanism (2). Similarly, staphostatin B, a cysteine protease inhibitor from Staphylococcus aureus, binds in a substrate-like manner in the active site of staphopain cysteine proteases. However, staphostatin B has a glycine PI residue, which adopts a backbone conformation that seems to prevent nucleophilic attack of the scissiie bond (3). 

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