Substrate specificity of recombinant human renal renin: effect of histidine in the P2 subsite on pH dependence.
Steady-state kinetic analysis of human renin demonstrates the histidine proximal to the substrate scissile peptide bond contributes to the unique specificity and pH dependence of this aspartyl protease. Recombinant human renal renin purified from mammalian cell culture appears to be indistinguishable from renin isolated from human kidney with respect to specific activity (1000 Goldblatt units/mg). Recombinant renin contains carbohydrate covalently attached to asparagines at positions 5 and 75 (renin numbering) and disulfide linkages at Cys-51/Cys-58, Cys-217/Cys-221, and Cys-259/Cys-296. Renin pH dependence was evaluated between pH 4.0 and 8.0 by using a synthetic substrate identical with the amino terminus of porcine angiotensinogen (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu*Leu-Val-Tyr-Ser, where the asterisk indicates the scissile peptide bond and the proximal histidine is in italics) and an analogous tetradecapeptide where the proximal histidine was substituted with glutamine. Comparison of the pH profiles shows the catalytic efficiency (V/Km) and maximal velocity (V) of renin are greater above pH 6.5 with the substrate containing histidine proximal to the scissile peptide bond, but below pH 5.0 these parameters are greater with the glutamine substrate analogue. Solvent isotope effects show that proton transfer contributes to the rate-limiting step in catalysis with both substrates and that the proximal histidine does not serve as a base in the catalytic mechanism. Molecular modeling indicates the substrate histidine could hydrogen bond to Asp-226 of the enzyme (renin numbering), thus perturbing the ionization of the catalytic aspartyl groups (Asp-38 and Asp-226).(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Biological Sciences, Monsanto Company, Chesterfield, Missouri 63198.
D W
DW
Green
S
S
Aykent
J K
JK
Gierse
M E
ME
Zupec
D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D004792:Enzyme Precursors / Q000235:genetics; D006639:Histidine; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008958:Models, Molecular; D011485:Protein Binding; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012083:Renin / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012084:Renin-Angiotensin System; D013379:Substrate Specificity; D014162:Transfection
D004792:Enzyme Precursors; D011994:Recombinant Proteins; D006639:Histidine; D012083:Renin