A single mutation at Tyr¹⁴³ of human S-adenosylhomocysteine hydrolase renders the enzyme thermosensitive and affects the oxidation state of bound cofactor nicotinamide–adenine dinucleotide

Recently, we have described the first human case of AdoHcyase (S-adenosylhomocysteine hydrolase) deficiency. Two point mutations in the AdoHcyase gene, the missense mutation p.Y143C (AdoHcyase in which Tyr¹⁴³ is replaced by cysteine) and the truncation mutation p.W112stop (AdoHcyase in which Trp¹¹² is replaced by opal stop codon) were identified [Barić, Fumić, Glenn, Ćuk, Schulze, Finkelstein, James, Mejaški-Bošnjak, Pažanin, Pogribny et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 4234–4239]. To elucidate the molecular and catalytic properties of AdoHcyase, we have made recombinant wild-type and mutant p.Y143C (AdoHcyase in which Tyr¹⁴³ is replaced by cysteine) enzymes for a comparative analysis. The catalytic rates of p.Y143C protein in the directions of S-adenosylhomocysteine synthesis or hydrolysis are decreased from 65% to 75%. Further, the oxidation states of coenzyme NAD differ between mutant and wild-type protein, with an increased NADH accumulation in the mutant p.Y143C enzyme of 88% NADH (wild-type contains 18% NADH). Quantitative binding of NAD is not affected. Native polyacrylamide gel electrophoresis showed, that mutant p.Y143C subunits are able to form the tetrameric complex as is the wild-type enzyme. CD analysis showed that the p.Y143C mutation renders the recombinant protein thermosensitive, with an unfolding temperature significantly reduced by 7 °C compared with wild-type protein. Change of Glu¹¹⁵ to lysine in wild-type protein causes a change in thermosensitivity almost identical with that found in the p.Y143C enzyme, indicating that the thermosensitivity is due to a missing hydrogen bond between Tyr¹⁴³ and Glu¹¹⁵. We emphasize involvement of this particular hydrogen bond for subunit folding and/or holoenyzme stability. In summary, a single mutation in the AdoHcyase affecting both the oxidation state of bound co-factor NAD and enzyme stability is present in a human with AdoHcyase deficiency.