The Role for Glutamic Acid at Position 196 in Human Hypoxanthine Phosphoribosyltransferase (HPRT) as Investigated Using Site-Directed Mutagenesis

• Published in: Nucleosides, nucleotides & nucleic acids Vol. 27; no. 6-7; pp. 894 - 899
• Main Authors: Canyuk, B., E-Wan, A., Keawwijit, W., Nualnoi, T., Sirisatean, L., Tansakul, P., Tanthana, C.
• Format: Journal Article
• Language: English
• Published: United States Taylor & Francis Group 01.06.2008
• Subjects: Catalysis; Dimerization; E196; functional role; Glutamic Acid - genetics; Glutamic Acid - metabolism; glutamic acid 196; Guanosine Monophosphate - metabolism; Human HPRT; Humans; Hypoxanthine Phosphoribosyltransferase - chemistry; Hypoxanthine Phosphoribosyltransferase - genetics; Hypoxanthine Phosphoribosyltransferase - metabolism; Kinetics; LND; Mutagenesis, Site-Directed; Mutant Proteins - chemistry; Mutant Proteins - genetics; Mutant Proteins - metabolism; Protein Structure, Quaternary; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Sepharose - metabolism
• ISSN: 1525-7770; 1532-2335
• DOI: 10.1080/15257770802146593

The crystal structure of human HPRT reveals the involvement of E196 side chain at the A-B dimer interface. Interference by valine substitution at this position (E196V), as identified in patients with Lesch-Nyhan disease, nearly abolishes enzymatic activity. Kinetic analysis of the active mutants (E196A, E196D, E196Q, and E196R) suggests that interaction between K68 and E196 side chains contributes to stabilization of cis-configuration during the catalytic cycle. The study also provides further insight into the role of A-B dimer interactions relating to K68 in the regulation of cis-trans isomerization that potentially governs the rate-limiting steps in the HPRT reaction.