Human pyridoxal kinase: overexpression and properties of the recombinant enzyme.
Pyridoxal kinase catalyses the phosphorylation of the vitamin B6. A human brain pyridoxal kinase cDNA was isolated, and the recombinant enzyme was overexpressed in E. coli as a fusion protein with maltose binding protein (MBP). Pure pyridoxal kinase exhibits a molecular mass of about 40 kDa when examined by SDS-PAGE and FPLC gel filtration. The recombinant enzyme is a monomer endowed with catalytic activity, indicating that the native quaternary structure of pyridoxal kinase is not a prerequisite for catalytic function. Zn2+ is the most effective divalent cation in the phosphorylation of pyridoxal, and the human enzyme has maximum catalytic activity in the narrow pH range of 5.5-6.0. The Km values for two substrates pyridoxal and ATP are 97 microM and 12 microM, respectively. In addition, the unfolding processes of the recombinant enzyme were monitored by circular dichroism. The values of the free energy change of unfolding (AGo = 1.2 kcal x mol(-1) x K(-1)) and the midpoint transition (1 M) suggested that the enzyme is more stable than ovine pyridoxal kinase against denaturation by guanidine hydrochloride. Intrinsic fluorescence spectra of the human enzyme from red-edge excitation and fluorescence quenching experiments showed that the tryptophanyl residues are not completely exposed and more accessible to neutral acrylamide than to the negatively charged iodide. The first complete set of catalytic and structural properties of human pyridoxal kinase provide valuable information for further biochemical studies on this enzyme.
Lee
H S
HS
Department of Biochemistry, Kyungpook National University, Taegu, Korea.
Moon
B J
BJ
Choi
S Y
SY
Kwon
O S
OS
D002412:Cations / Q000737:chemistry; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D011731:Pyridoxal Kinase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry; D013052:Spectrometry, X-Ray Emission; D014364:Tryptophan / Q000737:chemistry
D002412:Cations; D011994:Recombinant Proteins; D014364:Tryptophan; D011731:Pyridoxal Kinase