Transition thermolysin

Tetrhedral intermediate, 172 Thermodynamic cycles, 186 Thermolysin, zinc as cofactor for, 204 Thrombin, 170 Torsional potential, 111 Transition states, 41-42,44, 45,46, 88, 90-92 in amide hydrolysis, 219-221 oxyanion hole and, 181 stabilization of, 181,181 carbonium ion, 154,155,156-161, 167-169 for gas-phase reactions, 43... 

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... 

Fig. 3. Structure of the inhibitor phosphoramidon. The tetrahedral phosphorus atom binds at the active site of thermolysin and mimics the transition-state complex.

P. A. Bartlett and C. K. Marlowe, Phosphonamidates as transition-state analogue inhibitors of thermolysin, Biochemistry 1983, 22, 4618-4624. 

Figure 17.22. (a) Thermolysin-catalyzed hydrolysis of peptide analogs showing putative transition state, (b) phosphonamidate peptide analog, and (c) lluoroalkane peptide analog. 

Bartlett and Marlowe (153) designed a series of five phosphonamidate analogs of the peptide carbobenzoxy-Gly-Leu-X (X = NH2, Gly, Phe, Ala, Leu), where a -POz-NH- replaces the Gly-Leu peptide bond, and showed that these compounds were potent transition-state analog inhibitors of the zinc endopeptidase thermolysin. They also synthesized the corresponding phos-phonate analogs, where the -NH- (13A) is replaced by -O- (I3B). The... 

For the mechanistically related enzymes thermolysin and stromdysin, a strong case has been made supporting the hypothesis that the amine of leaving groups during turnover of peptide substrates is fully protonated in the transition state and thus requires no assistance from a general acid [71]. 

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. 

Figure 3 The rate-determining transition state in peptide hydrolysis by thermolysin from an AM1/AMBER QM/MM computation. The sticks and balls and sticks part correspond to the QM fragment. (See color plate at end of chapter.)...

An analysis of phosphonamidate and phosphonate transition-state analogs for the thermolysin reaction was reported (95, 96) and the data were interpreted to suggest that one hydrogen bond could contribute 4 kcal/mol binding to stabilize the formation of the tetrahedral transition state. A reevaluation was re-... 

The native OEE1 precursor was imported, processed and translocated to the thylakoid lumen (Rg. 1), as demonstrated by isolating thylakoids and showing that imported OEE1 protein was resistant to treatment with thermolysin but became sensitive subsequent to sonication. The translocation characteristics of OEE1 were demonstrated to be dependent on the OEE1 transit peptide. In the absence of the transit peptide, OEE1 neither imported, nor did it bind to isolated chloroplasts (data not shown). 

Figure 2. Phosphonamidate ( ), phosphinate (a), and phosphonate ( ) inhibitors of thermolysin as transition state analogs...

We have also looked at a series of tri- and tetrapeptide phosphonate inhibitors of carboxypeptidase A, with structural variation in the P2 and P3 residues.io Again, we see a good correlation between the Kj and Km/i cat values, supporting the view that the mechanisms of this enzyme and thermolysin are similar. This result is also consistent with the notion that a transition state analog motif that is good for one of these zinc peptidases should work the same way for the otho-. 

Bartlett, P.A. and Marlowe, C.K. (1983) Phosphonamidates as Transition State Analog Inhibitors of Thermolysin, Biochemistry, 22,4618-4624,... 

Holden, H.M., Tronrud, D ., Monzingo, A.F., Weaver, L.H. and Matthews, B.W. (1987) Slow- and Fast-Binding Inhibitors of Thermolysin Display Different Modes of Binding Crystallographic Analysis of Extended Phosphonamidate Transition-State Analogues, Biochemistry, 26, 8542-8553. 

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