Glucose phosphorylation. Interaction of a 50-amino acid peptide of yeast hexokinase with trinitrophenyl ATP.
A 50-amino acid peptide predicted by chemical modification studies of yeast hexokinase to contain an ATP-binding site has been synthesized and purified. The peptide, which includes residues from glutamate 78 at the NH2-terminal end to leucine 127 at the COOH-terminal, resides within the smaller of the two lobes found in the three-dimensional structure of yeast hexokinase. It is this region which has been reported recently to exhibit significant sequence homology with hexokinase types I and IV of higher eukaryotic cells and sequence homology with the active site of protein kinases. Similar to native yeast hexokinase, the 50-amino acid peptide interacts strongly with the fluorescent analog TNP-ATP [2',(3')-O-(2,4,6-trinitrophenyl)-adenosine-5'-triphosphate]. A 5-fold enhancement is observed when 8 microM peptide interacts with 20 microM TNP-ATP. The stoichiometry of binding is very close to 1 mol of TNP-ATP/mol peptide. Also, similar to native yeast hexokinase, the fluorescent enhancement observed upon TNP-ATP binding to the synthetic peptide is greater than that observed upon TNP-ADP binding. Finally, TNP-AMP exhibits a much lower fluorescent enhancement in the presence of hexokinase or the synthetic peptide. The additional findings that ATP can readily prevent TNP-ATP binding and that TNP-ATP can substitute for ATP as a weak substrate for hexokinase in the phosphorylation of glucose indicate that the synthetic peptide described here comprises part of the catalytic site.
Arora
K K
KK
Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Shenbagamurthi
P
P
Fanciulli
M
M
Pedersen
P L
PL
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology; D003196:Computer Graphics; D005947:Glucose / Q000378:metabolism; D006593:Hexokinase / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D010766:Phosphorylation; D011487:Protein Conformation; D011494:Protein Kinases / Q000235:genetics; D051381:Rats; D012689:Sequence Homology, Nucleic Acid
C009587:2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate; D010446:Peptide Fragments; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D006593:Hexokinase; D005947:Glucose