Enzymic properties of thermopsin.
The specificity of thermopsin, a thermostable acid protease from Sulfolobus acidocaldarius, was studied using oxidized insulin B chain as substrate followed by peptide isolation and identification. The following bonds were hydrolyzed: Leu-Val, Leu-Tyr, Phe-Phe, Phe-Tyr, and Tyr-Thr. Thus, the specificity of thermopsin is similar to that of pepsin, that is, it prefers large hydrophobic residues at both sides of the scissile bond. We confirmed this by the use of a synthetic substrate, Lys-Pro-Ala-Glu-Phe-p-nitro-phenylalanyl-Ala-Leu, which was cleaved by thermopsin between Phe and p-nitro-phenylalanyl. Using this substrate, enzyme inhibition and kinetic properties of thermopsin have been studied. Thermopsin optimally hydrolyzes this substrate at 75 degrees C and pH 2 with Km and kcat values under these conditions of 5.3 x 10(-5) M and 14.3 s-1, respectively. Pepstatin competitively inhibits thermopsin with a Ki of 2 x 10(-7) M. Other known aspartic protease inhibitors, diazoacetylnorleucine ethyl ester and 1,2-epoxy-3-(p-nitrophenoxy)propane inhibited thermopsin only slowly and with nonspecific reactions. Although thermopsin contains a single cysteine, iodoacetic acid and p-chloromercuric benzoate had no effect on activity. Mercuric chloride inhibited the enzyme, and the inhibition was reversible by mercaptoethanol. However, the enzyme was not labeled by [14C]iodoacetic acid either before or after sodium dodecyl sulfate denaturation. Thus, the thiol group is likely blocked, and the inhibition effect of mercuric ion is unrelated to the thiol group. These observations suggest that thermopsin has a different active site than the aspartic protease family but may have a similar transition state structure. The temperature dependence of Km and kcat was studied for thermopsin hydrolysis of the synthetic substrate between 26-78 degrees C. Both parameters increased with temperature, and the rise of kcat value was particularly sharp above 65 degrees C. Hydrolysis activity measured at high substrate concentration has a maximum at 76 degrees C, which is near the physiological temperature for the optimal growth of this organism. Thus, thermopsin appears to function best at high temperature and high substrate concentration. It may be utilized by the organism to response to the presence of high substrate concentration in the medium. Thermopsin is also competitively inhibited by urea, acetamide, and phenylalaninamide with Ki values of 0.5, 0.4, and 0.01 M, respectively.
Fusek
M
M
Laboratory of Protein Studies, Oklahoma Medical Research Foundation, Oklahoma City.
Lin
X L
XL
Tang
J
J
D000595:Amino Acid Sequence; D001105:Archaea / Q000201:enzymology*; D016282:Aspartic Acid Endopeptidases; D001419:Bacteria / Q000201:enzymology*; D010450:Endopeptidases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D007328:Insulin / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D010434:Pepsin A / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity; D013696:Temperature; D014508:Urea / Q000494:pharmacology
D007328:Insulin; D011480:Protease Inhibitors; D014508:Urea; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; D010434:Pepsin A; C062465:thermopsin