Characterization of the zinc sites in cobalamin-independent and cobalamin-dependent methionine synthase using zinc and selenium X-ray absorption spectroscopy.

Peariso K, Zhou ZS, Smith AE, Matthews RG, Penner-Hahn JE.

Biochemistry. 2001 Jan 30;40(4):987-93.


X-ray absorption spectroscopy has been used to investigate binding of selenohomocysteine to cobalamin-independent (MetE) and cobalamin-dependent (MetH) methionine synthase enzymes of Escherichia coli. We have shown previously [Peariso et al. (1998) J. Am. Chem. Soc. 120, 8410-8416] that the Zn sites in both enzymes show an increase in the number of sulfur ligands when homocysteine binds. The present data provide direct evidence that this change is due to coordination of the substrate to the Zn. Addition of L-selenohomocysteine to either MetE or the N-terminal fragment of MetH, MetH(2-649), causes changes in the zinc X-ray absorption near-edge structure that are remarkably similar to those observed following the addition of L-homocysteine. Zinc EXAFS spectra show that the addition of L-selenohomocysteine changes the coordination environment of the zinc in MetE from 2S + 2(N/O) to 2S + 1(N/O) + 1Se and in MetH(2-649) from 3S + 1(N/O) to 3S + 1Se. The Zn-S, Zn-Se, and Se-S bond distances determined from the zinc and selenium EXAFS data indicate that the zinc sites in substrate-bound MetE and MetH(2-649) both have an approximately tetrahedral geometry. The selenium edge energy for selenohomocysteine shifts to higher energy when binding to either methionine synthase enzyme, suggesting that there is a slight decrease in the effective charge of the selenium. Increases in the Zn-Cys bond distances upon selenohomocysteine binding together with identical magnitudes of the shifts to higher energy in the Se XANES spectra of MetE and MetH(2-649) suggest that the Lewis acidity of the Zn sites in these enzymes appears the same to the substrate and is electronically buffered by the Zn-Cys interaction.


L-Selenohomocysteine: one-step synthesis from L-selenomethionine and kinetic analysis as substrate for methionine synthases.

Zhou ZS, Smith AE, Matthews RG.

Bioorg Med Chem Lett. 2000 Nov 6;10(21):2471-5.


A single-step convenient synthesis of L-selenohomocysteine (SeHcy) from L-selenomethionine (SeMet) using sodium in liquid ammonia is described. Methionine synthases convert SeHcy to SeMet at rates comparable to their rates of conversion of L-homocysteine (Hcy) to L-methionine (Met). This study suggests that SeHcy generated from SeMet metabolism can be efficiently recycled to SeMet in mammals.


Identification of the zinc ligands in cobalamin-independent methionine synthase (MetE) from Escherichia coli.

Zhou ZS, Peariso K, Penner-Hahn JE, Matthews RG.

Biochemistry. 1999 Nov 30;38(48):15915-26.


Cobalamin-independent methionine synthase (MetE) from Escherichia coli catalyzes the transfer of a methyl group from methyltetrahydrofolate to homocysteine to form tetrahydrofolate and methionine. It contains 1 equiv of zinc that is essential for its catalytic activity. Extended X-ray absorption fine structure analysis of the zinc-binding site has suggested tetrahedral coordination with two sulfur (cysteine) and one nitrogen or oxygen ligands provided by the enzyme and an exchangeable oxygen or nitrogen ligand that is replaced by the homocysteine thiol group in the enzyme-substrate complex [Gonz�lez, J. C., Peariso, K., Penner-Hahn, J. E., and Matthews, R. G. (1996) Biochemistry 35, 12228-34]. Sequence alignment of MetE homologues shows that His641, Cys643, and Cys726 are the only conserved residues. We report here the construction, expression, and purification of the His641Gln, Cys643Ser, and Cys726Ser mutants of MetE. Each mutant displays significantly impaired activity and contains less than 1 equiv of zinc upon purification. Furthermore, each mutant binds zinc with lower binding affinity (K(a) approximately 10(14) M(-)(1)) compared to the wild-type enzyme (K(a) > 10(16) M(-)(1)). All the MetE mutants are able to bind homocysteine. X-ray absorption spectroscopy analysis of the zinc-binding sites in the mutants indicates that the four-coordinate zinc site is preserved but that the ligand sets are changed. Our results demonstrate that Cys643 and Cys726 are two of the zinc ligands in MetE from E. coli and suggest that His641 is a third endogenous ligand. The effects of the mutations on the specific activities of the mutant proteins suggest that zinc and homocysteine binding alone are not sufficient for activity; the chemical nature of the ligands is also a determining factor for catalytic activity in agreement with model studies of the alkylation of zinc-thiolate complexes.


An Antibody-Catalyzed Allylic Sulfoxide-Sulfenate Rearrangement.

Zhou ZS, Flohr A, Hilvert D.

J Org Chem. 1999 Oct 29;64(22):8334-8341.


Antibodies SZ-cis-39C11 and SZ-trans-28F8, which were elicited in response to N-aryl-3-methoxyphenyl proline derivatives, catalyze the [2,3]-sigmatropic rearrangement of allylic sulfoxides to sulfenates. Reduction of the sulfenates with dithiothreitol in situ yields allylic alcohols as the final product. The antibodies achieve rate accelerations in the range 10(2)-10(3) over background and exhibit distinctive hapten-dependent substrate specificity and enantio- and diastereoselectivity. Of particular note is the effective chirality transfer from the sulfoxide center to the product alcohol in the SZ-cis-39C11-catalyzed conversion of (Z)-2-(4-methoxyphenyl)-but-2-en-1-yl 4-nitrophenyl sulfoxide. These properties can be contrasted with those of bovine serum albumin (BSA) which accelerates the same reactions to a comparable extent but does not discriminate between substrate isomers. Partitioning of substrate from aqueous solution into the less polar environment of the protein pocket can account for much of the observed rate enhancement, whereas specific conformational constraints programmed by the haptens must orient the flexible substrate within the induced antibody-combining sites so as to favor certain reaction pathways over others. These studies thus expand the scope of antibody catalysis to an important new class of pericyclic reactions and illustrate how medium effects can be exploited together with conformational constraint to control reactivity and selectivity.


An Antibody-Catalyzed Selenoxide Elimination

Zhaohui S. Zhou, Ning Jiang, Donald Hilvert.

J. Am. Chem. Soc., 1997, 119 (15), pp 3623–3624 DOI: 10.1021/ja963748j
Publication Date (Web): April 16, 1997
Copyright © 1997 American Chemical Society


Three antibody catalysts for a [2,3]-sigmatropic elimination are described. The antibodies were elicited with N-aryl-3-methoxyphenyl proline derivs. and accelerate syn eliminations of primary and secondary alkyl arylselenoxides in aq. buffer to give olefins. In addn. to rate accelerations up to 2000-fold over background, the formation of significant yields of a normally disfavored cis olefin from a secondary selenoxide in the presence of one of the catalysts is notable. Substrate selectivity and kinetic data suggest that the obsd. catalytic effects are achieved through a combination of conformational constraint and medium effects. These studies thus expand the scope of antibody catalysis to an important new class of pericyclic reactions and provide novel protein catalysts for investigations of the factors that influence chem. reactivity.