Guanidine derivatives rescue the Arg418Ala mutation of Tritrichomonas foetus IMP dehydrogenase.
IMP dehydrogenase (IMPDH) catalyzes the oxidation of inosine 5'-monophosphate (IMP) to xanthosine 5'-monophosphate (XMP) and the reduction of NAD(+). The reaction involves formation of an E-XMP covalent intermediate; hydrolysis of the E-XMP intermediate is rate-limiting and requires the enzyme to adopt a closed conformation. Arg418 appears to act as the base that activates water for the hydrolysis reaction [Guillen-Schlippe, Y. V., and Hedstrom, L. (2005) Biochemistry 44, 11700-11707]. Deprotonation of Arg418 also stabilizes the closed conformation. Here we show that guanidine derivatives rescue the activity of the Arg418Ala variant. Amines and imidazole do not rescue. The rescue reaction appears to be saturable, with the values of K(R) ranging from 40 to 400 mM. The value of k(rescue) for the best rescue agents approaches the value of k(cat) for the reaction of the wild-type enzyme. Guanidine derivatives also rescue the activity of the Arg418Ala/Tyr419Phe variant. Multiple-inhibitor experiments suggest that the guanidine derivatives do not restore the equilibrium between open and closed conformations. Therefore, rescue agents must accelerate the hydrolysis of the E-XMP intermediate. The rate of the rescue reaction increases with an increase in pH, consistent with the hypothesis that the reaction involves neutral guanidine. A solvent D(2)O isotope effect is observed at low concentrations of the rescue agent, consistent with rate-limiting transfer of a proton from water. The value of k(cat) (rescue)/K(R)(base) correlates with the pK(a) of the guanidine derivative (Bronsted coefficient beta approximately 1). These results suggest that proton transfer from water to guanidine is almost complete in the transition state.
Guillén Schlippe
Yollete V
YV
Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02454, USA.
Hedstrom
Lizbeth
L
D000409:Alanine / Q000235:genetics*; D000818:Animals; D001120:Arginine / Q000235:genetics*; D019791:Guanidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D007168:IMP Dehydrogenase / Q000235:genetics*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D016835:Tritrichomonas foetus / Q000201:enzymology*
D001120:Arginine; D007168:IMP Dehydrogenase; D019791:Guanidine; D000409:Alanine