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Ternary complex structure of human HGPRTase, PRPP, Mg2+, and the inhibitor HPP reveals the involvement of the flexible loop in substrate binding

Published online by Cambridge University Press:  01 May 1999

GANESARATNAM K. BALENDIRAN ,
JOSÉ A. MOLINA ,
YIMING XU ,
JAN TORRES-MARTINEZ ,
ROBERT STEVENS ,
PAMELA J. FOCIA ,
ANN E. EAKIN ,
JAMES C. SACCHETTINI  and
SYDNEY P. CRAIG
GANESARATNAM K. BALENDIRAN
Affiliation:
Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843-2128
JOSÉ A. MOLINA
Affiliation:
Department of Biochemistry, University of Puerto Rico–Medical Sciences Campus, San Juan, Puerto Rico 00936 Present address: University of Puerto Rico, Cayey, Puerto Rico 00736.
YIMING XU
Affiliation:
Department of Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania 19140 Present address: Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461.
JAN TORRES-MARTINEZ
Affiliation:
Laboratory of Molecular Parasitology and Drug Design, School of Pharmacy, CB#7360, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599
ROBERT STEVENS
Affiliation:
Mass Spectrometry Facility, Section of Biochemical Genetics, Duke University Medical Center, Research Triangle Park, North Carolina 27709
PAMELA J. FOCIA
Affiliation:
Laboratory of Molecular Parasitology and Drug Design, School of Pharmacy, CB#7360, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599
ANN E. EAKIN
Affiliation:
Laboratory of Molecular Parasitology and Drug Design, School of Pharmacy, CB#7360, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599
JAMES C. SACCHETTINI
Affiliation:
Department of Biochemistry and Biophysics, Texas A&M University, College Station, Texas 77843-2128
SYDNEY P. CRAIG
Affiliation:
Laboratory of Molecular Parasitology and Drug Design, School of Pharmacy, CB#7360, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599
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Abstract

Site-directed mutagenesis was used to replace Lys68 of the human hypoxanthine phosphoribosyltransferase (HGPRTase) with alanine to exploit this less reactive form of the enzyme to gain additional insights into the structure activity relationship of HGPRTase. Although this substitution resulted in only a minimal (one- to threefold) increase in the Km values for binding pyrophosphate or phosphoribosylpyrophosphate, the catalytic efficiencies (kcat/Km) of the forward and reverse reactions were more severely reduced (6- to 30-fold), and the mutant enzyme showed positive cooperativity in binding of α-d-5-phosphoribosyl-1-pyrophosphate (PRPP) and nucleotide. The K68A form of the human HGPRTase was cocrystallized with 7-hydroxy [4,3-d] pyrazolo pyrimidine (HPP) and Mg PRPP, and the refined structure reported. The PRPP molecule built into the [(Fo − Fc)φcalc] electron density shows atomic interactions between the Mg PRPP and enzyme residues in the pyrophosphate binding domain as well as in a long flexible loop (residues Leu101 to Gly111) that closes over the active site. Loop closure reveals the functional roles for the conserved SY dipeptide of the loop as well as the molecular basis for one form of gouty arthritis (S103R). In addition, the closed loop conformation provides structural information relevant to the mechanism of catalysis in human HGPRTase.

Keywords

birth defectsinhibitor designPRPPPRTasepurine analograpid quench experimentssteady-state kinetic studies
Type
Research Article
Information
Protein Science , Volume 8 , Issue 5 , May 1999 , pp. 1023 - 1031
Copyright
1999 The Protein Society

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