Stopped-Flow Studies of Adenosylcobalamin Homolysis

-visible spectrum of the coenzyme changes significantly upon homolysis of the CooC bond AdoCbl is pink, a characteristic of the 6-coordinate Co(III) atom, whereas Cbl(II) is yellow-brown due to the presence of the 5-coordinate Co(II) species. This property provides a convenient and sensitive spectroscopic probe with which to monitor the enzyme-catalyzed homolysis of AdoCbl, the key step which results in the generation of free radicals. Thus, for several enzymes the rate of AdoCbl homolysis has been measured by stopped flow spectroscopy. 

B12-dependent ribonucleotide reductase was the first enzyme for which the rate of AdoCbl homolysis was examined. Pioneering studies by Blakely and co-workers established that a homolytic mechanism was indeed operating, as opposed to heterolytic cleavage to give Co(I), and that the rate of homolysis (k= 38s ) was kinetically competent (Tamao and Blakely, 1973). Subsequently, the kinetic competency of CooC cleavage in ethanolamine deaminase was established (Hollaway et al., 1978), and, as discussed below, homolysis has been found to be far more rapid than turnover for all the enzymes so far examined. 

FIGURE 16. Concerted and kinetically coupled mechanisms for CooC bond homolysis and hydrogen atom abstraction, illustrated for the reaction catalyzed by glutamate mutase. Either mechanism could give rise to the deuterium isotope effects observed in pre-steady state stopped flow experiments. 

A similar result has been observed with methylmalonyl-CoA mutase (Padmakumar and Banerjee, 1997). When this enzyme was reacted with prateo-methylmalonyl-CoA, homolysis of AdoCbl was almost complete within the deadtime of the stopped flow spectrometer. However, with (ds-methyl)-methylmalonyl-CoA homolysis is much slower and the isotope effeet is estimated to be at least 20. This suggests that coupling between CooC bond eleavage and substrate hydrogen abstraction is likely to be a general phenomenon. 

The most comprehensive set of kinetic studies on AdoCbl homolysis have been performed with AdoCbl-dependent ribonucleotide reductase by Stubbe and coworkers (Licht et al., 1999a Licht et al., 1999b). In this enzyme, AdoCbl is used to generate a thiyl radical on a cysteine residue it is this thiyl radical that abstracts hydrogen from the 3-position of the ribonucleotide to faeilitate reduction at C-2. In the presence of dGTP, an allosteric activator of the enzyme, the enzyme catalyzes the reversible cleavage of AdoCbl and formation of thiyl radical in the absence of substrate. This partial reaction proceeds rapidly enough for it to be mechanistically relevant and provides a system to study AdoCbl homolysis which is both simple and amenable to detailed kinetic analysis. 

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