Functional roles for amino acids in active site loop II of a hypoxanthine phosphoribosyltransferase.
A flexible loop of amino acids (loop II) closes over the active site of hypoxanthine phosphoribosyltransferase (HPRT) as the enzyme approaches the transition state [Biochemistry 37 (1998) 17120]. Formerly, the deletion of much of loop II from the HPRT of Trypanosoma cruzi resulted in a 2-3 order of magnitude reduction in k(cat) values with relatively modest changes in the Michaelis constants for substrates [Biochim. Biophys. Acta 1537 (2001) 63-70]. However, the contributions of individual loop II residues to catalysis remained poorly understood or have been disputed. Herein, saturation mutagenesis was used to generate relatively random sets of mutations in the 12 residues of active site loop II in the HPRT from T. cruzi and steady-state kinetics was used to investigate reactions catalyzed by the mutants. The results of analyses of 18 different mutations in an evolutionarily invariant Ser-Tyr dipeptide are consistent with interactions, between main chain nitrogen atoms of these residues and the O1A atom of phosphoribosylpyrophosphate (PRPP) or pyrophosphate (PPi), being essential for efficient enzyme chemistry. The results of analyses of 55 mutations in the nine other amino acids in loop II are inconsistent with these residues participating directly in enzyme chemistry, but are consistent with several of their side chains influencing loop flexibility and folding, as well as the efficiency for nucleotide formation relative to pyrophosphorolysis.
Medrano
Francisco J
FJ
Department of Molecular and Cell Biology, University of Connecticut, Laboratory of Molecular Parasitology and Drug Design, Storrs, CT, USA.
Wenck
Mary A
MA
Eakin
Ann E
AE
Craig
Sydney P
SP
D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D012694:Serine / Q000378:metabolism; D013329:Structure-Activity Relationship; D014349:Trypanosoma cruzi / Q000201:enzymology / Q000235:genetics; D014443:Tyrosine / Q000378:metabolism
D014443:Tyrosine; D012694:Serine; D007041:Hypoxanthine Phosphoribosyltransferase