Kinetics of the Inhibition of Aspartate Aminotransferase Isozymes by dl‐Glyceraldehyde 3‐Phosphate

Abstract

The inhibitor dissociation constants and types of inhibition of the isozymes of aspartate aminotransferase from rat liver by the time-dependent inhibitor DL-glyceraldehyde 3-phosphate were determined from residual enzyme activity after equilibration of each isozyme with the inhibitor and one substrate in the preliminary incubation mixtures.The results showed that the inhibition of the cationic isozyme was completely competitive with respect to a-ketoglutarate and completely noncompetitive with respect to L-aspartate. The pyridoxamine form ofthe cationic isozyme was approximately 10 times more sensitive to inhibition by DL-glyceraldehyde 3-phosphate (Ki = 0.084 mM) than the pyridoxal form (Ki = 0.98 mM). Inhibition of the anionic isozyme was mixed partially competitive-partially noncompetitive with respect to a-ketoglutarate and partially noncompetitive with respect to L-aspartate. The pyridoxamine form of the anionic isozyme was approximately 5 times more sensitive to inhibition by DL-glyceraldehyde 3-phosphate (Ki = 0.39 mM) than the pyridoxal form (Ki = 1.9 mM). Both forms of the cationic isozyme were more sensitive t o inhibition than the corresponding forms of the anionic isozyme.The degree to which inhibition of the anionic isozyme was partial was assessed by determining the interaction constants a and /3. These were a = 6.9 and /3 = 0.32, and indimted that the anionic isozyme, when fully combined with DL-glyceraldehyde 3-phosphate, retained 32 ", , of its catalytic capacity. The cationic isozyme was completely inhibited with a = 00 and /3 = 0.Comparison of the Ki values for the pyridoxamine and pyridoxal forms of the isozymes of aspartate amhotransferase with the concentrations of D-glyceraldehyde %phosphate reported for perfused liver and for incubated erythrocytes suggests that inhibition may occur in vivo.I n a previous communication [i], we demonstrated that DL-glyceraldehyde 3-phosphate is a time-dependent inhibitor of the cationic and anionic isozymes of aspartate aminotransferase purified from rat liver. Regulation of the activity of these isozymes has been implicated in the control of gluconeogenesis in vivo [1-51. I n the present study, the inhibitor dissociation constants and types of inhibition have been determined in order to evaluate the possibility of inhibition of the isozymes of aspartate aminotransferase in vivo by the glycolytic intermediate, D-glyceraldehyde 3-phosphate. Since the inhibition is time-dependent [i], the Ki values and the types of inhibition with respect to the substrates a-ketoglutarate and L-aspartate were determined from Unuaual Abbreviations. m-Glyceraldehyde 3-phosphate, glyceraldehyde-3-P ; pyridoxal 5'-phosphate, pyridoxal-5-P ; a-ketoglutarate, ketoglutarate or Kg ; L-aspartate, aspartate or Asp.Enzymes. Aspartate aminotransferase or L-aspartate : 2-oxoglutarate aminotransferase (EC 2.6.1.1). 249measurements of residual enzyme activity after equilibration of each isozyme with various concentrations of inhibitor and one substrate. Rate equations were derived for thes...

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“…Data were linearized using a Dixon plot of inverse initial steady-state velocities (1/V 0 ) versus inhibitor concentration [I]. K i app was determined by dividing the value for the y intercept by the slope of the line (25). The data were corrected to account for NCF (K m ϭ 11 Ϯ 1 M) for the ␤-lactamase using equation 1:…”

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“…Crude extracts, avibactam, and nitrocefin were mixed manually, and the initial velocity was monitored. Inverse initial steady-state velocities (1/ v 0 ) versus inhibitor concentration ( I ) were plotted, and the IC 50 values were determined by dividing the value for the y -intercept by the slope of line …”

“…Inverse initial steady-state velocities (1/v 0 ) versus inhibitor concentration (I) were plotted, and the IC 50 values were determined by dividing the value for the y-intercept by the slope of line. 32 The apparent K i (K i-app ) values were determined as described above for IC 50 values except using purified PenA1 or the R220A variant. Also, the data were corrected to account for the use of nitrocefin as a reporter substrate (PenA1 K m = 147 μM and R220A variant K m = 193 μM) via eq 1.…”