0162-0134/00/$ - see front matter q2000 Elsevier Science Inc. Al l rights reserved. PIIS0162-0134 ( 9 9 ) 00224-X Monday Mar 06 11:12 AM StyleTag -- Journal: JIB(Journal of Inorganic Biochemistry) Article:6318 www.elsevier.nl /locate/ jinorgbio Journal of Inorganic Biochemistry 78 (2000)149–160 Hydrogen abstraction from thiols by adenosyl radicals: chemical precedent for thiyl radical formation, the first catalytic step in ribonucleoside triphosphate reductase from Lactobacillus leichmannii Jeanne M. Sirovatka, Richard G. Finke * Chemistry Department, Colorado State University, Ft. Collins, CO 80523, USA Received 7 July 1999; received in revised form 2 November 1999; accepted 23 November 1999 Abstract Aqueous solutions of adenosylcobalamin ( AdoCbl)were thermolyzed with excess b-mercaptoethanol under anaerobic conditions. The product studies reveal that ;90% Co–C bond homolysis occurs, to yield Co(II)cobalamin, 59-deoxyadenosine, and the disulfide product fr om the combination of two HOCH 2 CH 2 S x radic als, 2,29-dithi odieth anol; there is also ;10% Co–C bond het ero lys is, yie ldi ng Co(III)cobalamin, adenine, and 2,3-dihydroxy-4-pentenal. The kinetic studies show there is a first-order dependence on AdoCbl and zero- order depende nce on thiol under the higher [RSH]experimental conditions used, consistent with the rate-determining step at high [RSH] being the generation of Ado x . The kinetic results require that, in enzyme-free AdoCbl solution, adenosyl radical (Ado x )isformed as a disc re te intermediate which then abstracts H x from the added thiol. The activation parameters for Co–C bond homolysis in the presence of thiol trap are the same within experimental error as the activation parameters for Co–C bond homolysis without trap, D H‡ obs s29(2)kcal mol y1 and DS‡ obs sy1(5)e.u. The results, in comparison to the rate of Co–C bond homolysis in ribonucleoside triphosphate reductase (RTPR), rev eal that RTPR accelerates Co–C bond cleavage in AdoCbl by ;10 10"1 . The recent literature evidence bearing on the exact mechanism of RTPR enzymic cleavage of the Co–C bond of AdoCbl is briefly discussed, notably the fact that this mechanism is presently controversial, but does involve at least coupled (and possibly concerted)Co–C cleavage, –S–H cleavage, and C–H (Ado–H)formation steps. q2000 Elsevier Science Inc. All rights reserved. Keywords:Hydrogen abstraction; Thiols; Adenosyl radicals; Ribonucleoside triphosphate reductase; Coenzyme B 12 ; Adenosylcobalamin Fig. 1. Coenzyme B 12 , also known as adenosylcobalamin ( AdoCbl ), and the schematic representation of AdoCbl used in the schemes herein. 1. Introduction Ribonucleotide reductases catalyze the transformation ofribonu cleoti des to deoxyribonuc leotid es in the rate-d etermin - ing step of DNA biosynthesis [1]; hence, all living cells require a ribonucleotide reductase[2,3]. There ar e curren tly fou r cla sse s of ribo nuc leotide red uct ase s, det ermine d by the ir enzyma tic cofact or [4]: Cl as sI co nta ins a diferri c cl uster and an obs erv abl e tyro syl rad ica l; Cla ss II contai ns coe nzy me B 12 (adenosylcobalamin; AdoCbl, Fig. 1), and is the class ofinterest to the present work; Class III contains a FeS cluster and an observ able glycyl radical; and the less studied Class IV enzymes, which are postulated to contain a dimanganese cluster and a tyrosyl radical. Relevant to the present work is the fact that all of the ribonucleo tide reduc tases are propos ed * Corres pon din g aut hor. Fax : q1-970-491-1801; e-mail : rfinke @ lamar.colostate.edu
