title: Active site and remote contributions to catalysis in methylthioadenosine nucleosidases.
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.
authors: †Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, United States.
authors: Keisha
authors: K
authors: Thomas
authors: †Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, United States.
authors: Scott A
authors: SA
authors: Cameron
authors: †Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, United States.
authors: Steven C
authors: SC
authors: Almo
authors: †Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, United States.
authors: Emmanuel S
authors: ES
authors: Burgos
authors: ‡The Ferrier Research Institute, Victoria University of Wellington, Wellington, New Zealand.
authors: Shivali A
authors: SA
authors: Gulab
authors: †Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, United States.
authors: Vern L
authors: VL
authors: Schramm
mesh_terms: D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000409:Alanine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution; D002384:Catalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D009699:N-Glycosyl Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013873:Thionucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014633:Valine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014734:Vibrio cholerae / Q000201:enzymology* / Q000235:genetics
chemical_list: D029968:Escherichia coli Proteins; D013873:Thionucleotides; C002480:methylthio-ADP; D000244:Adenosine Diphosphate; D009699:N-Glycosyl Hydrolases; C505576:5'-methylthioadenosine nucleosidase, E coli; D014633:Valine; D000409:Alanine