title: Femtosecond dynamics coupled to chemical barrier crossing in a Born-Oppenheimer enzyme.
abstract: Contributions of fast (femtosecond) dynamic motion to barrier crossing at enzyme catalytic sites is in dispute. Human purine nucleoside phosphorylase (PNP) forms a ribocation-like transition state in the phosphorolysis of purine nucleosides and fast protein motions have been proposed to participate in barrier crossing. In the present study, (13)C-, (15)N-, (2)H-labeled human PNP (heavy PNP) was expressed, purified to homogeneity, and shown to exhibit a 9.9% increase in molecular mass relative to its unlabeled counterpart (light PNP). Kinetic isotope effects and steady-state kinetic parameters were indistinguishable for both enzymes, indicating that transition-state structure, equilibrium binding steps, and the rate of product release were not affected by increased protein mass. Single-turnover rate constants were slowed for heavy PNP, demonstrating reduced probability of chemical barrier crossing from enzyme-bound substrates to enzyme-bound products. In a second, independent method to probe barrier crossing, heavy PNP exhibited decreased forward commitment factors, also revealing mass-dependent decreased probability for barrier crossing. Increased atomic mass in human PNP alters bond vibrational modes on the femtosecond time scale and reduces on-enzyme chemical barrier crossing. This study demonstrates coupling of enzymatic bond vibrations on the femtosecond time scale to barrier crossing.
authors: Department of Biochemistry, Albert Einstein College of Medicine of Yeshiva University, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
authors: Rafael G
authors: RG
authors: Silva
authors: Andrew S
authors: AS
authors: Murkin
authors: Vern L
authors: VL
authors: Schramm
mesh_terms: D001665:Binding Sites; D001671:Biochemistry / Q000379:methods; D002384:Catalysis; D004798:Enzymes / Q000737:chemistry; D006801:Humans; D007288:Inosine / Q000737:chemistry; D007700:Kinetics; D008956:Models, Chemical; D009038:Motion; D011485:Protein Binding; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry; D011683:Purine-Nucleoside Phosphorylase / Q000737:chemistry*; D055672:Static Electricity; D013379:Substrate Specificity
chemical_list: D004798:Enzymes; D011506:Proteins; D007288:Inosine; D011683:Purine-Nucleoside Phosphorylase