Structural and catalytic properties of a deletion derivative (delta 133-157) of Escherichia coli adenylate kinase.
Escherichia coli adenylate kinase (AKe) as well as the enzyme from yeast and mitochondria differs from the muscle cytosolic variant (AK1) by an insertion of 25 amino acid residues that are missing in AK1. The extra sequence, highly homologous in "large" size variants, is situated between residues 133 and 157 in AKe. Removal of 25 codons in the corresponding adk gene resulted in expression of a modified form of adenylate kinase (delta 133-157 AKe) which still conserved 7% of the maximal activity of the wild-type protein. The apparent Km for nucleotide substrates was increased by a factor of 4.6 (ADP), 23 (ATP) or 43 (AMP) in delta 133-157 AKe when compared with the wild-type enzyme. The secondary structure of delta 133-157 AKe, as well as its thermal stability were very similar to the parent protein. However, the deleted protein was much more sensitive than the wild-type enzyme to inactivation by trypsin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of trypsin digested delta 133-157 AKe revealed accumulation of several well defined fragments which were not observed in the case of wild-type enzyme. We conclude that the additional sequence, although necessary for expression of full activity in AKe, is not critical for catalysis. It is perhaps responsible for interaction of enzyme with other cellular components although a different mechanism of water shielding for large and small size variants of AK can be also envisaged.
Rose
T
T
Unité de Biochimie des Régulations Cellulaires, Institut Pasteur, Paris, France.
Brune
M
M
Wittinghofer
A
A
Le Blay
K
K
Surewicz
W K
WK
Mantsch
H H
HH
Bârzu
O
O
Gilles
A M
AM
D000263:Adenylate Kinase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002152:Calorimetry, Differential Scanning; D002851:Chromatography, High Pressure Liquid; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005798:Genes, Bacterial; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology; D010446:Peptide Fragments / Q000302:isolation & purification; D010957:Plasmids; D011487:Protein Conformation; D011817:Rabbits
D006146:Guanidines; D010446:Peptide Fragments; D000263:Adenylate Kinase; D019791:Guanidine