Role of protein conformational mobility in enzyme catalysis: acylation of alpha-chymotrypsin by specific peptide substrates.
To probe the mechanistic origins of convex Eyring plots that have been observed for alpha-chymotrypsin (alpha-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 alpha-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 15N-KIE for alkaline hydrolysis of p-nitroacetanilide. In 0.002 and 2 N hydroxide (30 degrees C), 15N-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 alpha-CT-catalyzed hydrolysis of Suc-Phe-pNA, 15N-KIE is on kc/Km 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 degrees 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, 15N-KIE does not vary over the temperature range 5-45 degrees 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 15N-KIE may point to a unique transition state for this process.
Hengge
Alvan C
AC
Department of Chemistry and Biochemistry, Utah State University, 0300 Old Main Hill, Logan, Utah 84322, USA.
Stein
Ross L
RL
D000083:Acetanilides / Q000737:chemistry; D000215:Acylation; D000577:Amides / Q000737:chemistry; D000813:Anilides / Q000737:chemistry; D000818:Animals; D002384:Catalysis; D002417:Cattle; D002918:Chymotrypsin / Q000737:chemistry* / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D009587:Nitrogen Isotopes / Q000737:chemistry; D009590:Nitrogen Radioisotopes / Q000737:chemistry; D009842:Oligopeptides / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D013379:Substrate Specificity; D013696:Temperature
D000083:Acetanilides; D000577:Amides; D000813:Anilides; D009587:Nitrogen Isotopes; D009590:Nitrogen Radioisotopes; D009842:Oligopeptides; D002918:Chymotrypsin; C117608:alpha-chymotrypsin