Mechanistic Studies on AdoCbl-dependent Glutamate Mutase.

Abstract

Adenosylcobalamin (AdoCbl) serves as a source of free radicals for a group of enzymes that catalyze unusual rearrangements involving hydrogen atom migration and proceed through a mechanism involving carbon-based free radical intermediates. Despite more than 50 years of detailed studies on AdoCbl-dependent enzymes, how enzymes catalyze homolysis of the cobalt-carbon bond of the coenzyme and stabilize the resulting free radicals is not well understood. AdoCbl-dependent glutamate mutase is a one of the simplest enzymes in which radical chemistry is employed. To better understand how the structure of the substrate influences the energetics of radical formation, the reaction of glutamate mutase with a glutamate analogue, 2-thiolglutarate has been investigated. 2-Thiolglutarate mimics the reaction with the natural substrate, glutamate however, abstraction of hydrogen from 2-thiolglutarate appears to be irreversible. This is most likely because upon forming the thioglutaryl radical, fragmentation occurs immediately to form the stable thioglycolyl radical which is too stable for the reaction to proceed toward the rearranged product due to delocalization of the unpaired electron on sulfur. Intrinsic deuterium KIE for hydrogen transfer between substrate and AdoCbl in glutamate mutase was measured by a novel experiment, employing intra-molecular isotope competition with a regio-specifically mono-deuterated substrate followed by analysis by high-resolution mass spectrometry. The intrinsic KIE in glutamate mutase was 7 ± 0.4 at 0 °C. This method was applied to investigate hydrogen tunneling effects in glutamate mutase. Temperature dependence studies on deuterium kinetic isotope effects in glutamate mutase between – 2.5 °C and 7.5 °C found the apparent isotope effects on AH/AD ~0.03 and ∆Ea(D-H) = 2.8 ± 0.5 kcal mol-1. This result demonstrates that hydrogen is extensively tunneling whereas deuterium is not. This is similar to the situation found for other AdoCbl-dependent systems studied previously, however deuterium KIE in glutamate mutase is much smaller. The reduced KIE in glutamate mutase can be explained based on previous secondary KIE studies which suggested coupling of the motions of the primary and secondary hydrogens combined with tunneling.