Abstract
Abnormal lysyl residues can be detected in aspartate transaminase by following the rate of reaction of amino groups with KN14CO and the rate of enzymatic inactivation. Peptide isolation subsequent to carbamylation of the apoenzyme produces a peptide which is absent in the carbamylated holoenzyme. The composition of the carbamylated peptide matches that of a tryptic peptide containing the active site Lys-258. The holoenzyme retains full catalytic activity after carbamylation of its NH2-terminal alanine and lysyl residues other than Lys-258, which is protected by aldimine formation with pyridoxal phosphate. Apoenzyme prepared from KNCO-treated holoenzyme (apoenzyme') is susceptible to further carbamylation at Lys-258 with irreversible loss of catalytic activity. Carbamylation of the active site lysyl residue is 25 to 50 times more rapid than that of the other 18 lysyl residues of aspartate transaminase. The kinetics of inactivation by KNCO at different pH values served to determine the pH-independent second order rate constant (k) and the pK of the amino group of Lys-258. These values are pK = 7.98 +/- 0.08 and k = 146 +/- 5 M-1S-1, which are similar to the values determined for carbamylation of the NH2- terminal groups of human hemoglobin (Garner, M. H., Bogardt, R. A., and Gurd, E. R. N. (1975) J. Biol. Chem. 250, 4398-4404). The pK value for Lys-258 is as low as that for a group in the active site region which can perturb a 19F nuclear magnetic resonance probe inserted into that region (Martinez-Carrion, M., Slebe, J. C., Boettcher, B., and Relimpio, A. M. (1976) J. Biol. Chem. 251, 1853-1858). Apoenzyme carbamylated at Lys-258 can accept pyridoxal phosphate at the active site even though no Schiff base in formed. Furthermore, this active site carbamylated holoenzyme will form spectroscopically detectable enzyme-substrate complexes with amino acids. The complexes slowly convert to species with absorbance identical with that of enzyme in the pyridoxamine phosphate form.
Highlights
The holoenzyme retains full catalytic activity after carbamylation of its NHz-terminal alanine and lysyl residues other than Lys-258, which is protected by aldimine formation with pyridoxal phosphate
Aspartate Transaminase by Cyanate-The susceptibility of the aspartate transaminase to inactivation by reaction with 0.2 M KNCO depends on the presence or absence of co-enzyme
Holoenzyme with the active site lysyl residue blocked by pyridoxal-P, as an internal aldimine, is functionally unaffected by the cyanate treatment
Summary
The holoenzyme retains full catalytic activity after carbamylation of its NHz-terminal alanine and lysyl residues other than Lys-258, which is protected by aldimine formation with pyridoxal phosphate. The kinetics of inactivation by KNCO at different pH values served to determine the pH-independent second order rate constant (k) and the pK of the amino group of Lys-258. These values are pK = 7.98 & 0.08 and k = 146 * 5 M-k’, which are similar to the values determined for carbamylation of the NH,-. Apoenzyme carbamylated at Lys-258 can accept pyridoxal phosphate at the active site even though no Schiff base is formed. Furtherpore, this active site carbamylated holoenzyme will form spectroscopically detectable enzyme.substrate complexes with amino acids. Several proteins have shown unusually reactive lysyl residues with anomalous pK values which can be as low as 5.9 for oxalacetate decarboxylase [4] and 7.70 * 0.3 in glutamate dehydrogenase [7]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
