Abstract
GTP cyclohydrolase II catalyzes the conversion of GTP into a mixture of 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate (Compound 2), formate, and pyrophosphate. Moreover, GMP was recently shown to be formed as a minor product. The major product (Compound 2) serves as the first committed intermediate in the biosynthesis of the vitamin, riboflavin. Numerous pathogenic microorganisms are absolutely dependent on endogenous synthesis of riboflavin. The enzymes of this pathway are therefore potential drug targets, and mechanistic studies appear relevant for development of bactericidal inhibitors. Pre-steady state quenched flow analysis of GTP cyclohydrolase II shows the rate-determining step to be located at the beginning of the reaction sequence catalyzed by the enzyme. Thus, GTP is consumed at a rate constant of 0.064 s(-1), and the reaction product, Compound 2, is formed at an apparent rate constant of 0.062 s(-1). Stopped flow experiments monitored by multiwavelength photometry are well in line with these data. 2-Amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone triphosphate can serve as substrate for GTP cyclohydrolase II but does not fulfill the criteria for a kinetically competent intermediate. A hypothetical reaction mechanism involves the slow formation of a phosphoguanosyl derivative of the enzyme under release of pyrophosphate. The covalently bound phosphoguanosyl moiety is proposed to undergo rapid hydrolytic release of formate from the imidazole ring and/or hydrolytic cleavage of the phosphodiester bond.
Highlights
GTP cyclohydrolase II catalyzes the conversion of GTP into a mixture of 2,5-diamino-6-ribosylamino-4(3H)pyrimidinone 5-phosphate (Compound 2), formate, and pyrophosphate
Unable to absorb the vitamin from the environment because of the absence of an appropriate uptake system; (ii) GTP cyclohydrolase II is absent in animals that are dependent on exogenous supply of vitamin
The GTP cyclohydrolases catalyzing the initial steps in the biosynthesis of folate and unconjugated pteridines and of riboflavin are slow catalysts with turnover numbers in the range of 1 to 5 per min and per subunit
Summary
Numerous pathogenic microorganisms are absolutely dependent on endogenous synthesis of riboflavin The enzymes of this pathway are potential drug targets, and mechanistic studies appear relevant for development of bactericidal inhibitors. Pre-steady state quenched flow analysis of GTP cyclohydrolase II shows the rate-determining step to be located at the beginning of the reaction sequence catalyzed by the enzyme. GTP cyclohydrolase II constitutes a potential antibiotics target for the following reasons: (i) numerous pathogenic bacteria, in particular Gram-negative organisms, are absolutely dependent on endogenous synthesis of the vitamin, because they are. GMP was recently identified as a genuine enzyme product that is formed by GTP cyclohydrolase II at a rate of about 10% as compared with Compound 2 [3]. This paper shows that the rate-determining step of GTP cyclohydrolase II is located at the start of the reaction sequence
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