1. A kinetic comparison of the homologous proteases astacin and meprin A
R L Wolz Arch Biochem Biophys. 1994 Apr;310(1):144-51. doi: 10.1006/abbi.1994.1150.
Astacin, a 23-kDa monomeric metalloprotease from the crayfish digestive tract, and meprin A, a 360-kDa tetrameric metalloprotease from the mouse kidney, are 30% identical in the amino acid sequence of their protease domains (Dumermuth et al., 1991, J. Biol. Chem. 266, 21381). The two were compared kinetically using a variety of substrates and inhibitors. Both enzymes degraded azocasein; meprin A had a twofold higher molar specific activity. Succinyl-Ala-Ala-Ala-p-nitroanilide was cleaved by both enzymes at the Ala-p-nitroanilide bond, indicating that in contrast to many metallopeptidases, peptidases of the astacin family are capable of arylamidolysis. Several peptides from a series of chromogenic and fluorogenic bradykinin analogs, originally designed for mapping the active site of meprin A, were found to be excellent substrates for astacin. Both enzymes cleaved most substrates at the site corresponding to the Phe5-Ser6 bond in native bradykinin (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg). The peptide 2ABz-Arg-Pro-Ile-Phe decreases Ser-Pro-Phe(4-nitro)-Arg was found to have the highest kcat/Km ratio for both peptidases (4.5 x 10(5) M-1 s-1 for meprin A and 2.7 x 10(6) M-1 s-1 for astacin). The two enzymes were found to have several other substrates in common, indicating that some of these peptides could be substrates for the other putative proteases in the astacin family. Acetyl-Arg-Pro-Gly-Tyr-NHOH, an inhibitor which likely binds to the S subsites, was found to act on both enzymes via a predominantly non-competitive mechanism, whereas NHOH-succinyl-Pro-Phe-Arg, which likely binds to the S' subsites, was competitive. Significant specificity differences between astacin and meprin A were seen in substrates and inhibitors with bulky groups in the P1' position. Substrates with Arg, Lys, or Phe in the P1' position were cleaved 10(3)-10(4) times faster by meprin A than by astacin. Actinonin, a naturally occurring peptide hydroxamate with a pentyl group in P1', was a very potent competitive inhibitor of meprin A (Ki = 1.35 x 10(-7) M), but had a 1000-fold weaker affinity for inhibition of astacin. These kinetic differences indicate that the S1' subsite is smaller in astacin than in meprin A and correlate with structural differences seen in the three-dimensional models of the two enzymes.
2. Hydrolysis of succinyl-trialanine p-nitroanilide by two enzymes associated with human high-density lipoproteins
H Maeda, S Kobori, H Uzawa Arch Biochem Biophys. 1983 Oct 15;226(2):629-35. doi: 10.1016/0003-9861(83)90332-6.
Succinyl-trialanine p-nitroanilide (Suc-Ala3-pNA), a synthetic substrate for the determination of elastase activity, was hydrolyzed in sequence by two enzymes that were found to be associated with human high-density lipoproteins. The enzymes involved in the sequence of reaction were separated by ion-exchange chromatography from apo-lipoprotein A-I and A-II, major apoproteins of high-density lipoproteins. One, designated as fraction MK, cleaves Suc-Ala3-pNA to succinyl-dialanine and alanine p-nitroanilide (Ala-pNA), and the other, designated as fraction U, cleaves Ala-pNA to alanine and p-nitroaniline. Fraction MK was inhibited by dithiothreitol, EDTA, and 1,10-phenanthroline, whereas fraction U was inhibited by 1,10-phenanthroline and bestatin. In addition to these findings, fraction MK also hydrolyzed 2,4-dinitrophenyl-prolyl-glutaminyl-glycyl-isoleucyl-alanyl-glycyl-glutaminyl- arginine (DNP-octapeptide), a specific substrate for the determination of vertebrate collagenase. Neither native elastin nor native collagen was hydrolyzed by a mixture of the two enzymes. Fraction U was very similar to aminopeptidase M with respect to its enzyme characteristics studied.
3. Specificity of pancreatic elastase with tripeptidyl-p-nitroanilide substrates
G C Szabó, M Pozsgay, R Gáspár, P Elödi Acta Biochim Biophys Acad Sci Hung. 1980;15(4):263-763.
1. The kinetic properties of pancreatic elastase (E.C. 3.4.21.11) were investigated with 33 tripeptidyl-p-nitroanilide substrates, and the Km, kcat and kcat/Km values were determined. 2. The individual contributions of the substrate side chains to the kinetic constants were evaluated by regression analysis. As a result of the additivity of the contributions, the kinetic parameters of any substrate constructed from the amino acid investigated can be predicted. 3. Suc-D-Phe-Pro-Ala-pNA was the best substrate, as far as the Km (Michaelis constant) is concerned. The compound was synthesized and assayed. An excellent correlation was observed between its calculated (14 microM) and experimentally determined (15 microM) Km values. The aldehyde derivatives of this substrate is a competitive inhibitor of elastase (Ki = 0.6 mM). 4. The contribution values of the best substrates permitted us to characterize the topography of subsites involved in the formation of the enzyme-substrate complex. This, in turn, led us to the conclusion that the S3-P3 interaction is relatively less important in the binding of good substrates.
