1. Phosphonamidates as transition-state analogue inhibitors of thermolysin
P A Bartlett, C K Marlowe Biochemistry. 1983 Sep 27;22(20):4618-24. doi: 10.1021/bi00289a002.
Six phosphorus-containing peptide analogues of the form Cbz-NHCH2PO2--L-Leu-Y (Y = D-Ala, NH2, Gly, L-Phe, L-Ala, L-Leu) have been prepared and evaluated as inhibitors of thermolysin. The Ki values for these compounds range from 1.7 microM to 9.1 nM and correlate well with the Km/kcat values for the corresponding peptide substrates [Morihara, K., & Tsuzuki, H. (1970) Eur. J. Biochem. 15, 374-380] but not with the Km values alone. The correlation noted between inhibitor Ki and substrate Km/kcat is the most extensive one of this type, providing strong evidence that the phosphonamidates are transition-state analogues and not simply multisubstrate ground-state analogues. Cbz-NH2CH2PO2--L-Leu-L-Leu (Ki = 9.1 nM) is the most potent inhibitor yet reported for thermolysin.
2. Inhibition of Streptomyces griseus metallo-endopeptidase II (SGMPII) by active-site-directed inhibitors
T Kumazaki, S Ishii, H Yokosawa J Biochem. 1994 Mar;115(3):532-5. doi: 10.1093/oxfordjournals.jbchem.a124370.
Inactivation of Streptomyces griseus metallo-endopeptidase II (SGMPII) by ClCH2CO-DL-(N-OH)Leu-OCH3 and by ClCH2CO-DL-(N-OH)Leu-Ala-Gly-NH2 was studied kinetically. These reagents cause irreversible inhibition of the enzyme in a pseudo-first order reaction, and the inhibition reaction exhibits saturation kinetics. The second-order rate constants for inactivation of SGMPII by ClCH2CO-DL-(N-OH)Leu-OCH3 and by ClCH2CO-DL-(N-OH)Leu-Ala-Gly-NH2 were measured to be 0.12 and 8.9 M-1.s-1, respectively. The order of affinities of metallo-endopeptidases towards these irreversible inhibitors is thermolysin > SGMPII > Pseudomonas aeruginosa elastase. A competitive inhibitor of SGMPII, L-Val-L-Trp, protects the enzyme against inactivation by ClCH2CO-DL-(N-OH)Leu-Ala-Gly-NH2 in a competitive manner. Furthermore, the pH profile of the inactivation closely resembles that for the hydrolysis of synthetic peptide substrates by the enzyme. These findings suggest that these reagents bind reversibly and react irreversibly at the active site of the enzyme.
3. Substrate and inhibitor studies of thermolysin-like neutral metalloendopeptidase from kidney membrane fractions. Comparison with bacterial thermolysin
M Pozsgay, C Michaud, M Liebman, M Orlowski Biochemistry. 1986 Mar 25;25(6):1292-9. doi: 10.1021/bi00354a015.
The inhibitory constants of a series of synthetic N-carboxymethyl peptide inhibitors and the kinetic parameters (Km, kcat, and kcat/Km) of a series of model synthetic substrates were determined for the membrane-bound kidney metalloendopeptidase isolated from rabbit kidney and compared with those of bacterial thermolysin. The two enzymes show striking similarities with respect to structural requirements for substrate binding to the hydrophobic pocket at the S1' subsite of the active site. Both enzymes showed the highest reaction rates with substrates having leucine residues in this position while phenylalanine residues gave the lowest Km. The two enzymes were also inhibited by the same N-carboxymethyl peptide inhibitors. Although the mammalian enzyme was more susceptible to inhibition than its bacterial counterpart, structural variations in the inhibitor molecules affected the inhibitory constants for both enzymes in a similar manner. The two enzymes differed significantly, however, with respect to the effect of structural changes in the P1 and P2' positions of the substrate on the kinetic parameters of the reaction. The mammalian enzyme showed the highest reaction rates and specificity constants with substrates having the sequence -Phe-Gly-Phe- or -Phe-Ala-Phe- in positions P2, P1, and P1', respectively, while the sequence -Ala-Phe-Phe- was the most favored by the bacterial enzyme. The sequence -Gly-Gly-Phe- as found in enkephalins was not favored by either of the enzymes. Of the substrates having an aminobenzoate group in the P2' position, the mammalian enzyme favored those with the carboxyl group in the meta position while the bacterial enzyme favored those with the carboxyl group in the para position.(ABSTRACT TRUNCATED AT 250 WORDS)
