Effect of pH on substrate and inhibitor kinetic constants of human liver alanine aminopeptidase. Evidence for two ionizable active center groups

Biochemistry
C W Garner, F J Behal

Abstract

The presence of at least two ionizable active center groups has been detected by a study of the effect of pH upon catalysis of hydrolysis of L-alanyl-beta-naphthylamide by human liver alanine aminopeptidase and upon the inhibition of hydrolysis by inhibitors and substrate analogs. Octanoic acid, octylamine, and peptide inhibitors have been found to be competitive inhibitors and are therefore thought to bind the active center. L-Phe was previously shown to bind the active center since it was found to be a competitive inhibitor of the hydrolysis of tripeptide substrates (Garner, C. W., and Behal, F. J. (1975), Biochemistry 14, 3208). A plot of pKm vs. pH for the substrate L-Ala-beta-naphthylamide showed that binding decreased below pH 5.9 and above 7.5, the points at which the theoretical curve undergoes an integral change in slope. These points are interpreted as the pKa either of substrate ionizable groups or binding-dependent enzyme active center groups. Similar plots of pKm vs. pH for L-alanyl-p-nitroanilide (as substrate) and pKi vs. pH for L-Leu-L-Leu-L-Leu and D-Leu-L-Tyr (as inhibitors) gave pairs fo pKa values of 5.8 and 7.4, 6.0 and 7.5, and 5.7 and 7.5, respectively. All the above substrates (and D-Leu-L-Tyr) have pKa ...Continue Reading

Citations

Aug 1, 1977·Archives of Biochemistry and Biophysics·C W Garner, F J Behal
Jan 1, 1980·Biochimica Et Biophysica Acta·J B McClellan, C W Garner
Jun 10, 1980·Clinica Chimica Acta; International Journal of Clinical Chemistry·W SidorowiczF J Behal
Nov 6, 1980·Clinica Chimica Acta; International Journal of Clinical Chemistry·W SidorowiczF J Behal
Feb 1, 1980·European Journal of Biochemistry·K HiwadaT Kokubu

Related Concepts

Abufne
Aminopeptidase
Hydrogen-Ion Concentration
Liver
Mathematics
Plasma Protein Binding Capacity
Structure-Activity Relationship
Thermodynamics

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