Substitution of the conserved Arg-Tyr dyad selectively disrupts the hydrolysis phase of the IMP dehydrogenase reaction.
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 k(cat) 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.
Guillén Schlippe
Yollete V
YV
Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02454, USA.
Riera
Thomas V
TV
Seyedsayamdost
Mohammad R
MR
Hedstrom
Lizbeth
L
D000244:Adenosine Diphosphate / Q000737:chemistry; D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001120:Arginine / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D002417:Cattle; D017124:Conserved Sequence / Q000235:genetics; D041961:Deuterium Exchange Measurement; D004791:Enzyme Inhibitors / Q000737:chemistry; D006868:Hydrolysis; D007168:IMP Dehydrogenase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009173:Mycophenolic Acid / Q000737:chemistry; D010649:Phenylalanine / Q000235:genetics; D017354:Point Mutation; D011485:Protein Binding / Q000235:genetics; D012254:Ribavirin / Q000031:analogs & derivatives* / Q000737:chemistry; D012263:Ribonucleosides / Q000737:chemistry; D012265:Ribonucleotides / Q000737:chemistry; D012997:Solvents; D013379:Substrate Specificity / Q000235:genetics; D016835:Tritrichomonas foetus / Q000201:enzymology / Q000235:genetics; D014443:Tyrosine / Q000235:genetics; D019820:Xanthine
D004791:Enzyme Inhibitors; D012263:Ribonucleosides; D012265:Ribonucleotides; D012997:Solvents; D019820:Xanthine; D014443:Tyrosine; D010649:Phenylalanine; D012254:Ribavirin; C010052:mizoribine; C011141:xanthosine monophosphate; D000244:Adenosine Diphosphate; D001120:Arginine; D007168:IMP Dehydrogenase; D009173:Mycophenolic Acid; D000409:Alanine; C033706:tiazofurin