Role of Protein Conformational Mobility in Enzyme Catalysis: Acylation of R-Chymotrypsin by Specific Peptide Substrates † Alvan C. Hengge ‡ and Ross L. Stein* ,#,§ Department of Chemistry and Biochemistry, Utah State UniVersity, 0300 Old Main Hill, Logan, Utah 84322, and Department of Neurology, HarVard Medical School, Laboratory for Drug DiscoVery in Neurodegeneration, HarVard Center for Neurodegeneration and Repair, 65 Landsdowne Street, Fourth Floor, Cambridge, Massachusetts 02139 ReceiVed October 15, 2003; ReVised Manuscript ReceiVed NoVember 12, 2003 ABSTRACT: To probe the mechanistic origins of convex Eyring plots that have been observed for R-chymotrypsin (R-CT)-catalyzed hydrolysis of specific p-nitroanilide substrates [Case, A., and Stein, R. L. (2003) Biochemistry 42, 3335-3348], we determined the temperature-dependence of 15 N-kinetic isotope effects for the R-CT-catalyzed hydrolysis of N-succinyl-Phe p-nitroanilide (Suc-Phe-pNA). To provide an interpretational context for these enzymatic isotope effects, we also determined 15 N-KIE for alkaline hydrolysis of p-nitroacetanilide. In 0.002 and 2 N hydroxide (30°C), 15 N-KIE values are 1.035 and 0.995 ((0.001), respectively, and are consistent with the reported [HO - ]-dependent change in rate-limiting step from leaving group departure from an anionic tetrahedral intermediate in dilute base, to hydroxide attack in concentrated base. For the R-CT-catalyzed hydrolysis of Suc-Phe-pNA, 15 N-KIE is on k c /K m and thus reflects structural features of transition states for all reaction steps up to and including acylation of the active site serine. The isotope effect at 35 °C is 1.014 ((0.001) and suggests that in the transition state for this reaction, departure of leaving group from the tetrahedral intermediate is well advanced. Significantly, 15 N-KIE does not vary over the temperature range 5-45 °C. This result eliminates one of the competing hypotheses for the convex Eyring plot observed for this reaction, that is, a temperature-dependent change in rate-limiting step within the chemical manifold of acylation, but supports a mechanism in which an isomerization of enzyme conformation is coupled to active site chemistry. We finally suggest that the near absolute temperature-independence of 15 N-KIE may point to a unique transition state for this pro- cess. It has been recognized for decades now that protein conformational isomerizations must play some role in enzyme catalysis. Examination of the literature reveals at least three ways in which conformational changes of the enzyme can participate in catalysis: (i) allowing the binding of substrate or departure of product, (ii) preorganizing active site residues for optimal interaction with substrates, and (iii) directly reducing activation energy by coupling to active site chemistry in catalytic transition states. In a recent study, Case and Stein (1) reported evidence supporting the third hypo- thesis for reactions of R-chymotrypsin (R-CT), 1 a well- studied member of the family of serine proteases. Serine proteases catalyze the hydrolysis of amide bonds of their protein and peptide substrates according to the three- step mechanism of Scheme 1. In the first step, substrate and enzyme combine to form the Michaelis complex. From within this complex, the hydroxyl of the active site serine attacks the carbonyl carbon of the amide bond of the substrate to generate an acyl-enzyme intermediate and liberate the first product. Finally, hydrolysis of the acyl-enzyme produces the † Portions of this work were supported by NIH Grant (GM47297) awarded to A.C.H. * To whom correspondence should be addressed at Laboratory for Drug Discovery in Neurodegeneration, Harvard Center for Neuro- degeneration and Repair, 65 Landsdowne Street, Fourth Floor, Cam- bridge, MA 02139. Phone: (617) 768-8651. Fax: (617) 768-8606. E-mail: rstein@rics.bwh.harvard.edu. ‡ Department of Chemistry and Biochemistry, Utah State University. # Department of Neurology, Harvard Medical School, § Laboratory for Drug Discovery in Neurodegeneration, Harvard Center for Neurodegeneration and Repair. 1 Abbreviations: Suc, N-succinyl; pNA, p-nitroanilide; PNAA, p-nitroacetanilide; R-CT, R-chrymotrypin; kE, kc/Km; 15 k, 15 N-isotope effect on the rate constant k; e.u., entropy unit or cal/mol. Scheme 1: Minimal Kinetic Mechanism for Serine Protease Catalysis 742 Biochemistry 2004, 43, 742-747 10.1021/bi030222k CCC: $27.50 © 2004 American Chemical Society Published on Web 12/31/2003