title: A novel engineered subtilisin BPN' lacking a low-barrier hydrogen bond in the catalytic triad.
abstract: The low-barrier hydrogen bond (LBHB) between the Asp and His residues of the catalytic triad in a serine protease was perturbed via the D32C mutation in subtilisin BPN' (Bacillus protease N'). This mutant enzyme catalyzes the hydrolysis of N-Suc-Ala-Ala-Pro-Phe-SBzl with a k(cat)/K(m) value that is only 8-fold reduced from that of the wild-type (WT) enzyme. The value of k(cat)/K(m) for the corresponding p-nitroanilide (pNA) substrate is only 50-fold lower than that of the WT enzyme (DeltaDeltaG++ = 2.2 kcal/mol). The pK(a) controlling the ascending limb of the pH versus k(cat)/K(m) profile is lowered from 7.01 (WT) to 6.53 (D32C), implying that any hydrogen bond replacing that between Asp32 and His64 of the WT enzyme most likely involves the neutral thiol rather than the thiolate form of Cys32. It is shown by viscosity variation that the reaction of WT subtilisin with N-Suc-Ala-Ala-Pro-Phe-SBzl is 50% (sucrose) to 100% (glycerol) diffusion-controlled, while that of the D32C construct is 29% (sucrose) to 76% (glycerol) diffusion-controlled. The low-field NMR resonance of 18 ppm that has been assigned to a proton shared by Asp32 and His64, and is considered diagnostic of a LBHB in the WT enzyme, is not present in D32C subtilisin. Thus, the LBHB is not an inherent requirement for substantial rate enhancement for subtilisin.
authors.affiliation: Department of Molecular and Cellular Biology, University of California, Berkeley, California 94720, USA.
authors.forename: J R
authors.initials: JR
authors.lastname: Stratton
authors.forename: J G
authors.initials: JG
authors.lastname: Pelton
authors.forename: J F
authors.initials: JF
authors.lastname: Kirsch
mesh_terms: D002384:Catalysis; D020134:Catalytic Domain; D003001:Cloning, Molecular; D005818:Genetic Engineering; D006860:Hydrogen Bonding; D007700:Kinetics; D013381:Subtilisins / Q000737:chemistry* / Q000235:genetics; D014783:Viscosity
chemical_list: D013381:Subtilisins