Ribonucleoside triphosphate reductase from Lactobacillus leichmannii: Kinetic evaluation of a series of adenosylcobalamin competitive inhibitors, [omega-(adenosin-5 '-O-yl)alkyl]cobalamins, which mimic the post Co-C homolysis intermediate

A series of [omega(adenosin-5'-O-yl)alkyl]cobalamins were examined for thei r inhibitory properties of ribonucleoside triphosphate reductase (RTPR) fro m Lactobacillus leichmannii in the presence of 5'-deoxyadenosylcobalamin (A doCbl, Coenzyme B-12). These AdoCbl analogs, in which oligomethylene chains (C-3-C-7) were inserted between the conin Co-atom and a 5'-O-atom of the a denosine moiety, were designed to probe the Co-C bond posthomolysis state i n AdoCbl-dependent enzymes, a state in which the Co and 5'-C distance is be lieved to be significantly increased. Experimentally, all five analogs were competitive inhibitors, with K-i in the range of 8-56 mu M. The [omega-(ad enosin-5'-O-yl)alkyl]cobalamin analog with C-5 methylene carbons was the st rongest inhibitor. This same pattern of inhibition, in which the C-5-analog is the strongest inhibitor, was previously observed in the AdoCbl-dependen t eliminase enzyme systems, diol dehydratase and glycerol dehydratase. Howe ver, in methylmalonyl CoA mutase, the strongest inhibition is by the C-6-an alog. This supports the hypothesis that the cobalamin posthomolysis interme diate in the eliminase enzymes differs from that in the mutase enzymes. The se findings led, in turn, to an examination of the Visible spectra of enzym e-bound cob(II)alamin in these two subclasses of AdoCbl-dependent enzymes. The results reveal an additional insight into the difference between the tw o classes: in the eliminases, the gamma-band of bound cob(II)alamin is shif ted from the 473 nm for free cob(II)alamin to longer wavelengths, 475-480 n m. However, in mutases, the gamma-band of bound cob(II)alamin is shifted to shorter wavelengths, 465-470 nm. Overall, the results (a) provide strong e vidence that two subclasses of AdoCbl-dependent enzymes exist, (b) give ins ights into the probable posthomolysis state in RTPR and other eliminases, a nd (c) identifies the C-5-analog as the tightest-binding analog for crystal lization and other biophysical studies. (C) 1999 Academic Press.