Role of Arg100 in the Active Site of Adenosylcobalamin-Dependent Glutamate Mutase † Li Xia, ‡ David P. Ballou, § and E. Neil G. Marsh* ,‡ Department of Chemistry and DiVision of Biophysics, UniVersity of Michigan, and Department of Biological Chemistry, UniVersity of Michigan Medical School, Ann Arbor, Michigan 48109-1055, USA ReceiVed September 29, 2003; ReVised Manuscript ReceiVed January 16, 2004 ABSTRACT: Arginine-100 is involved in recognizing the gamma carboxylate of the substrate in glutamate mutase. To investigate its role in substrate binding and catalysis, this residue was mutated to lysine, tyrosine, and methionine. The effect of these mutations was to reduce k cat by 120-320-fold and to increase K m(apparent) for glutamate by 13-22-fold; K m(apparent) for adenosylcobalamin is little changed by these mutations. Even at saturating substrate concentrations, no cob(II)alamin could be detected in the UV- visible spectra of the Arg100Tyr and Arg100Met mutants. However, in the Arg100Lys mutant cob(II)- alamin accumulated to concentrations similar to wild-type enzyme, which allowed the pre-steady-state kinetics of adenosylcobalamin homolysis to be investigated by stopped-flow spectroscopy. It was found that homolysis of the coenzyme is slower by an order of magnitude, compared with wild-type enzyme. Furthermore, glutamate binding is significantly weakened, so much so that the reaction exhibits second- order kinetics over the range of substrate concentrations used. The Arg100Lys mutant does not exhibit the very large deuterium isotope effects that are observed for homolysis of the coenzyme when the wild- type enzyme is reacted with deuterated substrates; this suggests that homolysis is slowed relative to hydrogen abstraction by this mutation. Glutamate mutase catalyzes the reversible isomerization of L-glutamate to L-threo-3-methylaspartate (1-5) and belongs to a group of adenosylcobalamin-dependent (AdoCbl, coenzyme B 12 ) 1 enzymes that catalyze unusual isomerization reactions that proceed by radical mechanisms; for recent reviews see refs 6-10. A mechanistic scheme for the enzyme is shown in Figure 1. The first step in the mechanism, common to all AdoCbl-dependent isomerases, is homolysis of the labile cobalt-carbon bond of AdoCbl to generate a 5′-deoxyadenosyl radical; this is followed by abstraction of hydrogen from the substrate to form 5′-deoxyadenosine (5′-dA) and a substrate radical. For glutamate mutase, the rearrangement of the C-4 radical of glutamate to form the methylaspartyl radical has been shown to proceed by a fragmentation-recombination mechanism with glycyl radical and acrylate as intermediates (11). The crystal structure of glutamate mutase has identified a number of residues that appear to make important hydrogen- bonding interactions with the substrate and coenzyme (12, 13). These include Arg149 and Arg66 that hydrogen bond to the R-carboxyl group of the substrate; Glu171, which makes a hydrogen bond to the amino group of the substrate, and Arg100 that forms a salt bridge with the γ-carboxylate, as illustrated in Figure 2. As part of our effort to determine how the enzyme promotes homolysis of the coenzyme and directs the reactive free radical intermediates formed toward productive catalysis, we have embarked upon a series of experiments to investigate the roles of various active site residues in the enzyme mechanism. Recently, we constructed and characterized several steri- cally and functionally conservative mutations of Glu171. The properties of the mutant proteins were consistent with the hypothesis that Glu171 acts as a general base that serves to deprotonate the amino group of the substrate during catalysis (14). Subsequently, pre-steady-state kinetic analysis of the Glu171Gln mutant revealed that several steps in the kinetic mechanism were altered by this mutation. Thus, substrate binding was weakened, and the apparent rate constants for homolysis of AdoCbl were significantly slowed compared with wild type. Furthermore, the mutation appeared to significantly reduce the large kinetic isotope effects on AdoCbl homolysis that are observed when the enzyme is reacted with deuterated substrates (15, 16). Here we describe experiments aimed at probing the role of Arg100 in substrate binding and catalysis. We have introduced several mutations at this position and investigated their effect on the steady-state kinetic properties of the enzyme, the ability of the enzyme to stabilize radical intermediates, and the effect of the mutations on the pre- steady-state kinetics of AdoCbl homolysis. MATERIALS AND METHODS Materials. Oligonucleotide synthesis and DNA sequencing were performed by the Biomedical Research Core facilities † This research has been supported by NIH Research Grants GM 59227 to E.N.G.M. and GM 20877 to D.P.B. * Correspondence should be addressed to this author at Department of Chemistry, University of Michigan, Ann Arbor, MI 48109-1055, USA. Tel (734) 763 6096. FAX: (734) 615 3790. E-mail: nmarsh@ umich.edu. § University of Michigan Medical School. ‡ Department of Chemistry, University of Michigan. 1 Abbreviations: AdoCbl, adenosylcobalamin; Cbl(II), cob(II)alamin; 5′-dA 5′-deoxyadenosine; GlmES, glutamate mutase fusion protein. 3238 Biochemistry 2004, 43, 3238-3245 10.1021/bi0357558 CCC: $27.50 © 2004 American Chemical Society Published on Web 02/27/2004