Active-Loop Dynamics within the Michaelis Complex of Lactate Dehydrogenase from Bacillus stearothermophilus.
Laser-induced temperature-jump relaxation spectroscopy was used to study the active site mobile-loop dynamics found in the binding of the NADH nucleotide cofactor and oxamate substrate mimic to lactate dehydrogenase in Bacillus stearothermophilus thermophilic bacteria (bsLDH). The kinetic data can be best described by a model in which NADH can bind only to the open-loop apoenzyme, oxamate can bind only to the bsLDH·NADH binary complex in the open-loop conformation, and oxamate binding is followed by closing of the active site loop preventing oxamate unbinding. The open and closed states of the loop are in dynamic equilibrium and interconvert on the submillisecond time scale. This interconversion strongly accelerates with an increase in temperature because of significant enthalpy barriers. Binding of NADH to bsLDH results in minor changes of the loop dynamics and does not shift the open-closed equilibrium, but binding of the oxamate substrate mimic shifts this equilibrium to the closed state. At high excess oxamate concentrations where all active sites are nearly saturated with the substrate mimic, all active site mobile loops are mainly closed. The observed active-loop dynamics for bsLDH is very similar to that previously observed for pig heart LDH.
Department of Biochemistry, Albert Einstein College of Medicine , Bronx, New York 10461, United States.
Beining
B
Nie
Department of Biochemistry, Albert Einstein College of Medicine , Bronx, New York 10461, United States.
Kara
K
Lodewyks
Department of Biochemistry, Albert Einstein College of Medicine , Bronx, New York 10461, United States.
Hua
H
Deng
Department of Chemistry, York College-CUNY, The CUNY Institute for Macromolecular Assemblies, and Ph.D. Programs in Chemistry and Biochemistry, The Graduate Center of the City University of New York , Jamaica, New York 11451, United States.
Ruel Z B
RZ
Desamero
Department of Biochemistry, Albert Einstein College of Medicine , Bronx, New York 10461, United States.
Robert
R
Callender
D000818:Animals; D001665:Binding Sites; D020134:Catalytic Domain; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism*; D009243:NAD / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D013379:Substrate Specificity; D013552:Swine; D013816:Thermodynamics
D009243:NAD; D007770:L-Lactate Dehydrogenase