Chemical modification and site-directed mutagenesis of cysteine residues in human placental S-adenosylhomocysteine hydrolase.
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 kcat values were only 7 and 12% of that of the wild-type enzyme, respectively, resulting in a calculated loss in binding energy (DeltaDeltaG) 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.
Department of Biochemistry, The University of Kansas, Lawrence, Kansas 66047, USA. borchardt@smissman.hbc.ukans.edu
C S
CS
Yuan
D B
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Ault-Riché
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Borchardt
D043383:Adenosylhomocysteinase; D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D006801:Humans; D006867:Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007460:Iodoacetamide / Q000494:pharmacology; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010920:Placenta / Q000201:enzymology*; D014357:Trypsin / Q000378:metabolism
D004228:Dithionitrobenzoic Acid; D006867:Hydrolases; D043383:Adenosylhomocysteinase; D014357:Trypsin; D007460:Iodoacetamide