Substrate specificities and kinetic properties of proteinase A from the yeast Saccharomyces cerevisiae and the development of a novel substrate.
The substrate specificities and kinetic properties of proteinase A, an intracellular aspartic proteinase from the yeast Saccharomyces cerevisiae, were determined using a series of synthetic chromogenic peptides with the general structure P5-P4-P3-P2-Phe-(NO2)Phe-P2'-P3' [P5, P4, P3, P2, P2', P3' are various amino acids; (NO2)Phe is p-nitro-L-phenylalanine]. The nature of the residues occupying the NH2-terminal region of the substrate had a strong influence on the kinetic constants. Among those tested, Ala-Pro-Ala-Lys-Phe-(NO2)-Phe-Arg-Leu had the best kinetic constants (Km = 0.012 mM, kcat = 14.4 s-1, kcat/Km = 1,200 M-1.s-1). Compared with such aspartic proteinases as pepsin, cathepsin D, and renin, the substrate specificity of proteinase A was unique. Based on these results, a novel fluorescent substrate, MOCAc-Ala-Pro-Ala-Lys-Phe-Phe-Arg-Leu-Lys(Dnp)-NH2, was developed for the sensitive measurement of proteinase A.
Kondo
H
H
Institute for Fundamental Research, Suntory Limited, Shimamoto-cho, Mishima-gun, Osaka, 618-0024, Japan. Hiroto_Kondo@suntory.or.jp
Shibano
Y
Y
Amachi
T
T
Cronin
N
N
Oda
K
K
Dunn
B M
BM
D019297:2,4-Dinitrophenol / Q000031:analogs & derivatives / Q000378:metabolism; D000595:Amino Acid Sequence; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism; D010434:Pepsin A / Q000378:metabolism; D012083:Renin / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013379:Substrate Specificity
D005456:Fluorescent Dyes; D009842:Oligopeptides; C114307:methyloxycarbonylacetyl-alanyl-prolyl-alanyl-lysyl-phenylalanyl-phenylalanyl-arginyl-leucyl-lysyl-(2,4-dinitrophenyl)-amide; D016282:Aspartic Acid Endopeptidases; D010434:Pepsin A; D012083:Renin; C022442:aspergillopepsin II; D019297:2,4-Dinitrophenol