Identification of lysyl residues at the AMP-binding site of biodegradative threonine deaminase from Escherichia coli.
The biodegradative threonine deaminase from Escherichia coli is activated allosterically by AMP. To identify the residues interacting with the phosphate group of AMP at the binding site, we used the affinity labeling reagent, adenosine diphosphopyridoxal (AP2-PL). In the absence of AMP, the enzyme formed the Schiff base with AP2-PL and Scatchard plot analysis showed a biphasic pattern, the respective Kd values for the high- and low-affinity binding phases being 20 and 110 microM. The former value is comparable to the Kd value of the enzyme for AMP. In the presence of AMP, the Schiff base formation was greatly reduced. Although the maximal activating effect of adenosine diphosphopyridoxine, a non-reactive derivative of AP2-PL, was about 13% of that of AMP, the half-saturation concentration was almost the same. These findings suggest that AP2-PL specifically labeled the lysyl residue(s) at the AMP-binding site of the enzyme. To identify the labeled residue(s), we reduced the modified enzyme with sodium borohydride, then cleaved it with cyanogen bromide and Achromobacter lyticus protease I. Reverse-phase HPLC was used to isolate two labeled peptides from the digest. Their amino acid compositions and sequences showed that Lys-111 and Lys-113 were labeled. We conclude that these two lysyl residues are located around the phosphate group of AMP at the allosteric regulation site of the enzyme.
Hirose
K
K
Department of Chemistry, Faculty of Science, Kyoto University.
Kawata
Y
Y
Yumoto
N
N
Tokushige
M
M
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000249:Adenosine Monophosphate / Q000378:metabolism*; D000345:Affinity Labels; D000495:Allosteric Site; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives*; D013913:Threonine Dehydratase / Q000737:chemistry / Q000378:metabolism*
D000345:Affinity Labels; D000249:Adenosine Monophosphate; D011732:Pyridoxal Phosphate; D000244:Adenosine Diphosphate; D013913:Threonine Dehydratase; C048236:pyridoxal 5'-diphospho-5'-adenosine; D008239:Lysine