Determination of kinetic constants for peptidyl prolyl cis-trans isomerases by an improved spectrophotometric assay.
The kinetic properties and substrate specificity of two well-characterized peptidyl prolyl cis-trans isomerases (PPIases), cyclophilin and the FK-506 binding protein (FKBP), have been previously examined [Fischer, G., Bang, H., Berger, E., & Schellenberger, A. (1984) Biochim. Biophys. Acta 791, 87-97; Harrison, R.K., & Stein, R.L. (1990) Biochemistry 29, 1684-1689; Albers, M.W., Walsh, C.T., & Schreiber, S. L. (1990) J. Org. Chem. 55, 4984-4986]. The chymotrypsin-coupled enzymatic assay employed in these studies suffers from two serious shortcomings. Due to the low equilibrium population of the X-cis-Pro-Phe-pNA isomer (the PPIase substrate), in conjunction with the low solubility of p-nitroaniline generated by chymotrypsin hydrolysis, substrate concentrations in the saturating region are not experimentally attainable. Secondly, the uncatalyzed cis-trans isomerization obscures the interpretation of the initial velocity. As a result of these limitations, the steady-state kinetic parameters (Km,Kcat) have not been determined. Here we introduce an improved version of the spectrophotometric assay and report for the first time the Michaelis constants and turnover numbers for both PPIases with established substrates. The improvements in the experimental conditions originate in a medium-induced increase in the equilibrium population of the cis X-Pro conformer and in conducting the assay at 0 degrees C to suppress the uncatalyzed thermal isomerization. In addition, we present a rigorous mathematical model of the spectrophotometric progress curves that accounts for the contributions of the residual background rate.(ABSTRACT TRUNCATED AT 250 WORDS)
Kofron
J L
JL
School of Pharmacy, University of Wisconsin, Madison 53706.
Kuzmic
P
P
Kishore
V
V
Colón-Bonilla
E
E
Rich
D H
DH
D000591:Amino Acid Isomerases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000814:Aniline Compounds / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002417:Cattle; D002918:Chymotrypsin / Q000494:pharmacology; D003524:Cyclosporins / Q000494:pharmacology*; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D019696:Peptidylprolyl Isomerase; D013053:Spectrophotometry; D013237:Stereoisomerism; D013379:Substrate Specificity / Q000187:drug effects; D013950:Thymus Gland / Q000201:enzymology
D000814:Aniline Compounds; D002352:Carrier Proteins; D003524:Cyclosporins; C008625:nitroaniline; D002918:Chymotrypsin; D000591:Amino Acid Isomerases; D019696:Peptidylprolyl Isomerase