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abstracts
One way for enzymes to affect reactions they catalyze is through transition state stabilization. Another factor to be considered is the contribution of substrate distortion, although it has been thoroughly described for only a few enzymes. We have a longstanding interest in the reaction mechanism of orotidine monophosphate decarboxylase (ODCase) and determined various crystal structures bound with distorted substrates at around 1.5 Å resolution. The enzyme is known as one of the most proficient enzymes, which accelerates the decarboxylation of orotidine 5'-monophosphate (OMP) to form uridine 5'-monophosphate (UMP) by 17 orders of magnitude. One argument against the contribution of substrate distortion to the ODCase reaction is the weak affinity of UMP. The distortions observed so far all appear at the C6-substituent of the pyrimidine ring, which corresponds to the carboxylate of OMP. Since the carboxylate is removed by the reaction, the product UMP should bind more tightly to ODCase than OMP, if the distortion of C6-substituent contributes to the catalysis. In order to investigate this inconsistency, we determined the crystal structure of ODCase with UMP at atomic resolution (1.03 Å). The structure showed an unfavorable interaction between UMP and the catalytic residue K72, an interaction considered to be absent in the OMP complex. Surface plasmon resonance analysis indicated that UMP binds stronger to the K72A mutant than to the wild-type enzyme by 5 orders of magnitude. These analyses invalidate the argument against a contribution of substrate distortion to ODCase catalysis. Finally, we estimated how much the distortion contributes to the catalysis using computational simulation methods. The results indicated that 10-15% decrease of the ΔΔG‡ value is contributed by substrate distortion.
abstracts
The TK2285 protein from a hyperthermopilic archaeon Thermococcus kodakarensis is a myo-inositol kinase. Only two myo-inositol kinases have been identified so far. One is the TK2285 protein and the other is an enzyme from Zea mays. Both of them synthesize myo-inositol monophosphate that shows enantiomerism. Because it is too difficult to discriminate enantiomers by NMR or chromatography analysis, it has not been identified which of the six hydroxyls is phosphorylated by these enzymes. Also, little is known about the substrate recognition of myo-inositol kinase, since only the unliganded crystal structure of TK2285 has been reported. In order to reveal the substrate-binding mechanism of myo-inositol kinase and identify the phosphorylated hydroxyl group of the product, we determined the crystal structures of TK2285 as the substrate-complex and the product-complex. The substrate-complex of TK2285 was prepared by using the TK2285, myo-inositol and AMP-PCP, and the products-complex was prepared by incubating the TK2285 with myo-inositol and ATP. The substrate-complex structure showed that all of the six hydroxyls of myo-inositol interacted with TK2285. This coincides with the fact that the Km value for myo-inositol is 100-1000 fold lower than those for other sugars. Also 3-hydroxyl group of myo-inositol, which the gamma-phosphate of AMP-PCP was nearest to, was thought to be phosphorylated by this enzyme. This was proved by the product-complex structure that had ADP and myo-inositol 3-phosphate. Site-directed mutagenesis and structure comparison with TK2285 homologs also provided information about the substrate-binding mechanism of myo-inositol kinase.
