mechanism of rate-limiting motions in enzyme function

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 104; no. 29; pp. 11981 - 11986
• Main Authors: Watt, Eric D, Shimada, Hiroko, Kovrigin, Evgenii L, Loria, J. Patrick
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 17-07-2007; National Acad Sciences
• Subjects: Active sites; Amino acids; Atoms; Binding sites; Biochemistry; Biological Sciences; Catalysis; Chemical equilibrium; Enzymes; Histidine - chemistry; Hydrogen bonds; Isotope effects; Isotopes; Kinetics; Movement; NMR; Nuclear magnetic resonance; Nuclear Magnetic Resonance, Biomolecular; Protein Structure, Secondary; Protons; Ribonuclease, Pancreatic - chemistry; Ribonuclease, Pancreatic - metabolism; Ribozymes; Solvents; Studies
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.0702551104

The ability to use conformational flexibility is a hallmark of enzyme function. Here we show that protein motions and catalytic activity in a RNase are coupled and display identical solvent isotope effects. Solution NMR relaxation experiments identify a cluster of residues, some distant from the active site, that are integral to this motion. These studies implicate a single residue, histidine-48, as the key modulator in coupling protein motion with enzyme function. Mutation of H48 to alanine results in loss of protein motion in the isotope-sensitive region of the enzyme. In addition, kcat decreases for this mutant and the kinetic solvent isotope effect on kcat, which was 2.0 in WT, is near unity in H48A. Despite being located 18 Å from the enzyme active site, H48 is essential in coordinating the motions involved in the rate-limiting enzymatic step. These studies have identified, of [almost equal to]160 potential exchangeable protons, a single site that is integral in the rate-limiting step in RNase A enzyme function.