Substrate specificity and mechanism from the structure of Pyrococcus furiosus galactokinase.
Galactokinase (GalK) catalyses the first step of the Leloir pathway of galactose metabolism, the ATP-dependent phosphorylation of galactose to galactose-1-phosphate. In man, defects in galactose metabolism can result in disorders with severe clinical consequences, and deficiencies in galactokinase have been linked with the development of cataracts within the first few months of life. The crystal structure of GalK from Pyrococcus furiosus in complex with MgADP and galactose has been determined to 2.9 A resolution to provide insights into the substrate specificity and catalytic mechanism of the enzyme. The structure consists of two domains with the active site in a cleft at the domain interface. Inspection of the substrate binding pocket identifies the amino acid residues involved in galactose and nucleotide binding and points to both structural and mechanistic similarities with other enzymes of the GHMP kinase superfamily to which GalK belongs. Comparison of the sequence of the Gal3p inducer protein, which is related to GalK and which forms part of the transcriptional activation of the GAL gene cluster in the yeast Saccharomyces cerevisiae, has led to an understanding of the molecular basis of galactose and nucleotide recognition. Finally, the structure has enabled us to further our understanding on the functional consequences of mutations in human GalK which cause galactosemia.
Krebs Institute, Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, UK.
Andrew
A
Hartley
Steven E
SE
Glynn
Vladimir
V
Barynin
Patrick J
PJ
Baker
Svetlana E
SE
Sedelnikova
Corné
C
Verhees
Daniel
D
de Geus
John
J
van der Oost
David J
DJ
Timson
Richard J
RJ
Reece
David W
DW
Rice
D000244:Adenosine Diphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D005686:Galactokinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005690:Galactose / Q000378:metabolism; D005693:Galactosemias / Q000201:enzymology / Q000235:genetics; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D017510:Protein Folding; D020027:Pyrococcus furiosus / Q000201:enzymology* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D055672:Static Electricity; D013379:Substrate Specificity; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics
C099289:Gal3 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D000244:Adenosine Diphosphate; D005686:Galactokinase; D005690:Galactose