Substitution of the Conserved Arg-Tyr Dyad Selectively Disrupts the Hydrolysis Phase of the IMP Dehydrogenase Reaction

Yollete V.Guillén Schlippe, Thomas V. Riera, Mohammad R. Seyedsayamdost, Lizbeth Hedstrom

Research output: Contribution to journalArticlepeer-review

28 Scopus citations

Abstract

Inosine 5′-monophosphate dehydrogenase (IMPDH) catalyzes the oxidation of IMP to XMP via the covalent E-XMP* intermediate (E-XMP*), with the concomitant reduction of NAD+. Hydrolysis of E-XMP* is rate-limiting, and the catalytic base required for this step has not been identified. An X-ray crystal structure of Tritrichomonas foetus IMPDH with mizoribine monophosphate (MZP) reveals a novel closed conformation in which a mobile flap occupies the NAD+/NADH site [Gan, L., Seyedsayamdost, M. R., Shuto, S., Matsuda, A., Petsko, G. A., and Hedstrom, L. (2003) Biochemistry 42, 857-863]. In this complex, a water molecule is coordinated between flap residues Arg418 and Tyr419 and MZP in a geometry that resembles the transition state for hydrolysis of E-XMP*, which suggests that the Arg418-Tyr419 dyad activates water. We constructed and characterized two point mutants, Arg418Ala and Tyr419Phe, to probe the role of the Arg418-Tyr419 dyad in the IMPDH reaction. Arg418Ala and Tyr419Phe decrease kcat by factors of 500 and 10, respectively, but have no effect on hydride transfer or NADH release. In addition, the mutants display increased solvent isotope effects and increased levels of steady-state accumulation of E-XMP*. Inhibitor analysis indicates that the mutations destabilize the closed conformation, but this effect can account for a decrease in k cat of no more than a factor of 2. These observations demonstrate that both the Arg418Ala and Tyr419Phe mutations selectively impair hydrolysis of E-XMP* by disrupting the chemical transformation. Moreover, since the effects of the Tyr419Phe mutation are comparatively small, these experiments suggest that Arg418 acts as the base to activate water.

Original languageEnglish (US)
Pages (from-to)4511-4521
Number of pages11
JournalBiochemistry
Volume43
Issue number15
DOIs
StatePublished - Apr 20 2004
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biochemistry

Fingerprint

Dive into the research topics of 'Substitution of the Conserved Arg-Tyr Dyad Selectively Disrupts the Hydrolysis Phase of the IMP Dehydrogenase Reaction'. Together they form a unique fingerprint.

Cite this