Ribonucleoside Triphosphate Reductase from Lactobacillus leichmannii: Kinetic Evaluation of a Series of Adenosylcobalamin Competitive Inhibitors, [ω-(Adenosin-5′-O-yl)alkyl]cobalamins, Which Mimic the Post Co-C Homolysis Intermediate

A series of [ω-(adenosin-5′-O-yl)alkyl]cobalamins were examined for their inhibitory properties of ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii in the presence of 5′-deoxyadenosylcobalamin (AdoCbl, Coenzyme B12). These AdoCbl analogs, in which oligomethylene chains (C3-C7) were inserted between the corrin Co-atom and a 5′-O-atom of the adenosine moiety, were designed to probe the Co-C bond posthomolysis state in AdoCbl-dependent enzymes, a state in which the Co and 5′-C distance is believed to be significantly increased. Experimentally, all five analogs were competitive inhibitors, with Ki in the range of 8–56 μM. The [ω-(adenosin-5′-O-yl)alkyl]cobalamin analog with C5 methylene carbons was the strongest inhibitor. This same pattern of inhibition, in which the C5-analog is the strongest inhibitor, was previously observed in the AdoCbl-dependent eliminase enzyme systems, diol dehydratase and glycerol dehydratase. However, in methylmalonyl CoA mutase, the strongest inhibition is by the C6-analog. This supports the hypothesis that the cobalamin posthomolysis intermediate in the eliminase enzymes differs from that in the mutase enzymes. These findings led, in turn, to an examination of the visible spectra of enzyme-bound cob(II)alamin in these two subclasses of AdoCbl-dependent enzymes. The results reveal an additional insight into the difference between the two classes: in the eliminases, the γ-band of bound cob(II)alamin is shifted from the 473 nm for free cob(II)alamin to longer wavelengths, 475–480 nm. However, in mutases, the γ-band of bound cob(II)alamin is shifted to shorter wavelengths, 465–470 nm. Overall, the results (a) provide strong evidence that two subclasses of AdoCbl-dependent enzymes exist, (b) give insights into the probable posthomolysis state in RTPR and other eliminases, and (c) identifies the C5-analog as the tightest-binding analog for crystallization and other biophysical studies.