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A single mutation at tyrosine (143) of human S-adenosylhomocysteine hydrolase renders the enzyme thermosensitive and effects the oxidation state of bound cofactor nicotinamide-adenine dinucleotide


Belužić, Robert; Ćuk, Mario; Pavkov, Tea; Fumić, Ksenija; Barić, Ivo; Mudd, Harvey S.; Jurak, Igor; Vugrek, Oliver
A single mutation at tyrosine (143) of human S-adenosylhomocysteine hydrolase renders the enzyme thermosensitive and effects the oxidation state of bound cofactor nicotinamide-adenine dinucleotide // Biochemical Journal, 400 (2006), 2; 245-253 (međunarodna recenzija, članak, znanstveni)


Naslov
A single mutation at tyrosine (143) of human S-adenosylhomocysteine hydrolase renders the enzyme thermosensitive and effects the oxidation state of bound cofactor nicotinamide-adenine dinucleotide

Autori
Belužić, Robert ; Ćuk, Mario ; Pavkov, Tea ; Fumić, Ksenija ; Barić, Ivo ; Mudd, Harvey S. ; Jurak, Igor ; Vugrek, Oliver

Izvornik
Biochemical Journal (0264-6021) 400 (2006), 2; 245-253

Vrsta, podvrsta i kategorija rada
Radovi u časopisima, članak, znanstveni

Ključne riječi
AdoHcyase deficiency; hydrogen bond; circular dichroism; DTNB; recombinant protein; in vitro mutagenesis

Sažetak
Recently, we have described the first human case of S-adenosylhomocysteine hydrolase deficiency. Two point mutations in the AdoHcyase gene, the missense mutation p.Y143C and the truncation mutation p.W112X were identified [Barić, Fumić, Glenn, Ćuk, Schulze, Finkelstein, Jill James, Mejaški-Bošnjak, Pažanin, Pogribny, Radoš, Sarnavka, Šćukanec-Špoljar, Allen, Stabler, Uzelac, Vugrek, Wagner, Zeisel, Mudd (2004) PNAS USA 101, 4234-4239]. To elucidate the molecular and catalytic properties of S-adenosylhomocysteine hydrolase, we have made recombinant wild-type and mutant p.Y143C enzymes for a comparative analysis. The catalytic rates of p.Y143C protein in the directions of S-adenosylhomocysteine synthesis or hydrolysis are decreased 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 wildtype enzyme. CD analysis showed that the p.Y143C mutation renders the recombinant protein thermosensitive, with an unfolding temperature significantly reduced by 7°C compared to wild-type protein. Change of E115 to L115 in wild-type protein causes a change in thermosensitivity almost identical to that found in the p.Y143C enzyme, indicating that the thermosensitivity is due to a missing hydrogen bond between Y143 and E115. We emphasize involvement of this particular hydrogen bond for subunit folding and/or holoenyzme stability. In summary, a single mutation in the S-adenosylhomocysteine hydrolase affecting both the oxidation state of bound co-factor NAD and enzyme stability is present in a human with S-adenosylhomocysteine hydrolase deficiency

Izvorni jezik
Engleski

Znanstvena područja
Biologija, Temeljne medicinske znanosti, Biotehnologija



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  • Current Contents Connect (CCC)
  • Web of Science Core Collection (WoSCC)
    • Science Citation Index Expanded (SCI-EXP)
    • SCI-EXP, SSCI i/ili A&HCI
  • Scopus
  • MEDLINE