Year: 2015
Type: article
Abstract: 5'-Methylthioadenosine/S-adenosyl-l-homocysteine nucleosidases (MTANs) catalyze the hydrolysis of 5'-methylthioadenosine to adenine and 5-methylthioribose. The amino acid sequences of the MTANs from Vibrio cholerae (VcMTAN) and Escherichia coli (EcMTAN) are 60% identical and 75% similar. Protein structure folds and kinetic properties are similar. However, binding of transition-state analogues is dominated by favorable entropy in VcMTAN and by enthalpy in EcMTAN. Catalytic sites of VcMTAN and EcMTAN in contact with reactants differ by two residues; Ala113 and Val153 in VcMTAN are Pro113 and Ile152, respectively, in EcMTAN. We mutated the VcMTAN catalytic site residues to match those of EcMTAN in anticipation of altering its properties toward EcMTAN. Inhibition of VcMTAN by transition-state analogues required filling both active sites of the homodimer. However, in the Val153Ile mutant or double mutants, transition-state analogue binding at one site caused complete inhibition. Therefore, a single amino acid, Val153, alters the catalytic site cooperativity in VcMTAN. The transition-state analogue affinity and thermodynamics in mutant VcMTAN became even more unlike those of EcMTAN, the opposite of expectations from catalytic site similarity; thus, catalytic site contacts in VcMTAN are unable to recapitulate the properties of EcMTAN. X-ray crystal structures of EcMTAN, VcMTAN, and a multiple-site mutant of VcMTAN most closely resembling EcMTAN in catalytic site contacts show no major protein conformational differences. The overall protein architectures of these closely related proteins are implicated in contributing to the catalytic site differences.
Source: Biochemistry
DOI: 10.1021/bi501487w
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
Funders: U.S. Department of Energy, Eli Lilly and Company, Price Family Foundation, National Institutes of Health, Office of Science, National Cancer Institute, National Institute of General Medical Sciences, National Center for Research Resources, Basic Energy Sciences, Biological and Environmental Research, Argonne National Laboratory
Awards: G102163711, G1026088249, G107546177, G1286251239, G1313983767, G1664757510, G1842308918, G2287611682, G2463063366, G262962599, G2857925935, G3075337988, Transition State Analysis of Enzymatic Reactions, Protein Dynamics in Enzymatic Catalysis, Macromolecular Crystallography at the National Synchrotron Light Source, Transition State Analysis of Enzymatic Reactions, G3981344285, G409777406, G426311843, G498139845, G521541070, G5387317844, G5607048534, G6120118751, G6848031779, G6918803902, Macromolecular Crystallography at the National Synchrotron Light Source, G7583137537, G7982793873, G9000035606, G9000135192, G9000234506, Cancer Center Support Grant, G969889393, G993149817
OpenAlex ID: W2331504047