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Volume 271, Number 45, Issue of November 8, 1996 pp. 28009-28016
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Chemical Modification and Site-directed Mutagenesis of Cysteine Residues in Human Placental S-Adenosylhomocysteine Hydrolase

(Received for publication, July 17, 1996, and in revised form, August 22, 1996)

From the Department of Biochemistry, The University of Kansas, Lawrence, Kansas 66047

Human placental S-adenosylhomocysteine (AdoHcy) hydrolase (EC 3.3.1.1) was inactivated by 5,5-dithiobis(2-nitrobenzoic acid) following pseudo-first-order kinetics. Modification of three of the 10 cysteine residues per enzyme subunit resulted in complete inactivation of the enzyme. The three modified cysteine residues were identified as Cys¹¹³, Cys¹⁹⁵, and Cys⁴²¹, respectively, by protein sequencing after modification with [1-¹⁴C]iodoacetamide. Of the three modifiable cysteines, Cys¹¹³ and Cys¹⁹⁵ could be protected from modification in the presence of the substrate adenosine (Ado), which also protected the enzyme from inactivation. On the other hand, Cys⁴²¹ was not protected by Ado, and modification of Cys⁴²¹ alone did not affect the enzyme activity. To verify whether some of these cysteine residues are important for the enzyme catalysis, these three cysteine residues were replaced by either serine or aspartic acid using site-directed mutagenesis. Mutants of both Cys¹¹³ (C113S and C113D) and Cys⁴²¹ (C421S and C421D) had enzyme activities similar to that of the wild-type enzyme, and only slight changes were observed in the steady-state kinetics measured in both the synthetic and hydrolytic directions. However, mutants of Cys¹⁹⁵ (C195D and C195S) displayed drastically reduced enzyme activities, and k_cat values were only 7 and 12% of that of the wild-type enzyme, respectively, resulting in a calculated loss in binding energy (G) of approximate 1 Kcal/mol. The Cys¹⁹⁵ mutants were capable of catalyzing both the 3-oxidative and 5-hydrolytic reactions, as evidenced by the reduction of E·NAD⁺ to NADH and formation of the 5-hydrolytic product when incubated with (E)-5,6-didehydro-6-deoxy-6-chlorohomoadenosine at rates comparable with those catalyzed by the wild-type enzyme. However, mutations of the Cys¹⁹⁵ severely altered the 3-reduction potential as evidenced by the drastic reduction in the rate of [2,8-³H]Ado release from the E^-NADH·[2,8-³H]3-keto-Ado complex. Circular dichroism studies of the Cys¹⁹⁵ mutants confirmed that the observed effects are not due to changes in secondary structure. These results suggested that the Cys¹⁹⁵ is involved in the catalytic center and may play an important role in maintaining the 3-reduction potential for effective release of the reaction products and regeneration of the active form (NAD⁺ form) of the enzyme; the Cys¹¹³ is located in or near the substrate binding site, but plays no role beneficial to the catalysis; and the Cys⁴²¹ is a nonessential residue, which also explains why Cys⁴²¹ does not occur in any other known AdoHcy hydrolases.

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