Is Arg418 the catalytic base required for the hydrolysis step of the IMP dehydrogenase reaction?
The first committed step of guanine nucleotide biosynthesis is the oxidation of inosine 5'-monophosphate (IMP) to xanthosine 5'-monophosphate (XMP) catalyzed by IMP dehydrogenase. The reaction involves the reduction of NAD(+) with the formation of a covalent enzyme intermediate (E-XMP). Hydrolysis of E-XMP requires the enzyme to adopt a closed conformation and is rate-limiting. Thr321, Arg418, and Tyr419 are candidates for the residue that activates water. The substitution of Thr321 has similar, but small, effects on both the hydride transfer and hydrolysis steps. This result suggests that Thr321 influences the reactivity of Cys319, either through a direct interaction or by stabilizing the structure of the active site loop. The hydrolysis of E-XMP is accelerated by the deprotonation of a residue with a pK(a) of approximately 8. A similar deprotonation stabilizes the closed conformation; this residue has a pK(a) of >or=6 in the closed conformation. The substitution of Tyr419 with Phe does not change the pH dependence of either the hydrolysis of E-XMP or the conformational change, which suggests that Tyr419 is not the residue that activates water. In contrast, the conformational change becomes pH-independent when Arg418 is substituted with Gln. Lys can replace the function of Arg418 in the hydrolysis reaction but does not stabilize the closed conformation. The simplest explanation for these observations is that Arg418 serves as the base that activates water in the IMPDH reaction.
Guillén Schlippe
Yollete V
YV
Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02454, USA.
Hedstrom
Lizbeth
L
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001120:Arginine / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007168:IMP Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D016835:Tritrichomonas foetus / Q000201:enzymology
D001120:Arginine; D007168:IMP Dehydrogenase