Inactivation of the Lactobacillus leichmannii ribonucleoside triphosphate reductase by 2'-chloro-2'-deoxyuridine 5'-triphosphate: stoichiometry of inactivation, site of inactivation, and mechanism of the protein chromophore formation

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Abstract
The ribonucleoside triphosphase reductate (RTPR) of Lactobacillus leichmannii is inactivated by the substrate analogue 2''-chloro-2''-deoxyuridine 5''-triphosphate (ClUTP). Inactivation is due to alkylation by 2-methylene-3(2H)-furanone, a decomposition product of the enzymic product 3''-keto-2''-deoxyuridine triphosphate. The former has been unambiguously identified as 2-[(ethylthio)methyl]-3(2H)-furanone, an ethanethiol trapped adduct, which is identical by 1H NMR spectroscopy with material synthesized chemically. Subsequent to rapid inactivation, a slow process occurs that results in formation of a new protein-associated chromophore absorbing maximally near 320 nm. The terminal stages of the inactivation have now been investigated in detail. The alkylation and inactivation stoichiometries were studied as a function of the ratio of ClUTP to enzyme. At high enzyme concentrations (0.1 mM), 1 equiv of [5''-3H]ClUTP resulted in 0.9 equiv of 3H bound to protein and 83% inactivation. The amount of labelling of RTPR increased with increasing ClUTP concentration up to the maximum of approximately 4 labels/RTPR, yet the degree of inactivation did not increase proportionally. This suggests that (1) RTPR may be inactivated by alkylation of a single site and (2) decomposition of 3''-keto-dUTP is not necessarily enzyme catalyzed. The formation of the new protein chromophore was also monitored during inactivation and found to reach its full extent upon the first alkylation. Thus, out of four alkylation sites, only one appears capable of undergoing the subsequent reaction to form the new chromophore. While chromophore formation was prevented by NaBH4 treatment, the chromophore itself is resistant to reduction. Model studies suggest that the new chromophore is due to addition of an amino group to the 5-position of enzyme-bound furanone, followed by ring opening and tautomerization to give a .beta.-aminoenone structure. When inactivation by [2''-3H]ClUTP is performed in the presence of NaBH4 in order to stabilize the label and the protein is treated with iodoacetamide and then digested with trypsin, three very closely eluting peptides are observed by high-performance liquid chromatography. Sequencing of the first of these peptides demonstrated that the label was present on one of the active-site thiols of RTPR [Lin, A., Ashley, G. W., and Stubbe, J. (1987) Biochemistry 26, 6905-6909].