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.
Skip Nav Destination
Article navigation
December 2006
-
Cover Image
Cover Image
- PDF Icon PDF LinkFront Matter
- PDF Icon PDF LinkTable of Contents
- PDF Icon PDF LinkEditorial Board
Research Article|
November 14 2006
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
Robert Belužić;
Robert Belužić
*Division of Molecular Medicine, Institute Ruđer Bošković, Bijenička 54, 10000 Zagreb, Croatia
Search for other works by this author on:
Mario Ćuk;
Mario Ćuk
†Department of Pediatrics, School of Medicine, University Hospital Center, 10000 Zagreb, Croatia
Search for other works by this author on:
Tea Pavkov;
Tea Pavkov
‡Institute of Chemistry, Structural Biology, Karl-Franzens-University, 8010 Graz, Austria
Search for other works by this author on:
Ksenija Fumić;
Ksenija Fumić
§Clinical Institute of Laboratory Diagnosis, University Hospital Center, Zagreb 10000, Croatia
Search for other works by this author on:
Ivo Barić;
Ivo Barić
†Department of Pediatrics, School of Medicine, University Hospital Center, 10000 Zagreb, Croatia
Search for other works by this author on:
S. Harvey Mudd;
S. Harvey Mudd
∥Laboratory of Molecular Biology, National Institute of Mental Health, Bethesda, MD 20892-9663, U.S.A.
Search for other works by this author on:
Igor Jurak;
Igor Jurak
¶Division of Viral Infections, Robert Koch Institute, D-10963 Berlin, Germany
Search for other works by this author on:
Oliver Vugrek
Oliver Vugrek
1
*Division of Molecular Medicine, Institute Ruđer Bošković, Bijenička 54, 10000 Zagreb, Croatia
1To whom correspondence should be addressed (email ovugrek@irb.hr).
Search for other works by this author on:
Publisher: Portland Press Ltd
Received:
May 22 2006
Revision Received:
July 14 2006
Accepted:
July 27 2006
Accepted Manuscript online:
July 27 2006
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2006
Biochem J (2006) 400 (2): 245–253.
Article history
Received:
May 22 2006
Revision Received:
July 14 2006
Accepted:
July 27 2006
Accepted Manuscript online:
July 27 2006
Citation
Robert Belužić, Mario Ćuk, Tea Pavkov, Ksenija Fumić, Ivo Barić, S. Harvey Mudd, Igor Jurak, Oliver Vugrek; 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. Biochem J 1 December 2006; 400 (2): 245–253. doi: https://doi.org/10.1042/BJ20060749
Download citation file:
Sign in
Don't already have an account? Register
Sign in to your personal account
You could not be signed in. Please check your email address / username and password and try again.
Captcha Validation Error. Please try again.