| Abstract: | Dihydropyrimidine amidohydrolase (DHPase) has been purified from bovine liver in large quantities. The amino acid composition has been determined by two independent sources. The N-terminal sequence is hydrophobic and includes pyroglutamate as the N-terminal residue. The enzyme isoelectric point is 5.2-5.4. The multiple bands and peaks from isoelectric focusing and chromatofocusing are due to deamidation of asparagine and glutamine residues. Thermal inactivation first order plots are non-linear, indicating an enzyme microheterogeneous population. Steady-state kinetics for hydrolysis and ring closure reactions demonstrate biphasic double reciprocal plots, due to either microheterogeneity of the enzyme population, negative cooperativity or substrate activation. The double reciprocal plots of chromatofocusing pools are biphasic, indicating that the enzyme microheterogeneity is not the cause of the biphasic kinetics. Downward curvature in Hill plots suggests negative cooperativity. When the enzyme is maximally activated with N-bromoacetamide, the double reciprocal plots are linear. This phenomenon may be due to the modification of a non-catalytic substrate binding site, which probably binds substrate after the active site is filled with high substrate concentrations, produces a conformational change and increases catalytic activity. The pH rate profiles indicate pK[subscript a] values of 6.0 for free enzyme and 5.2 for enzyme-substrate complex. Diethylpyrocarbonate (DEP), a chemical modifier fairly specific for histidine residues, only activates DHPase. Photooxidation with Rose Bengal and Methylene Blue show an activation process followed by complete inactivation. Amino acid analysis after treatment with both dyes indicates that two to three histidine residues are involved in both the activation and inactivation of DHPase. N-Bromoacetamide (NBA) also completely inactivates the enzyme following an activation that is enhanced by a competitive inhibitor, dihydroorotic acid. The amino acid analysis indicates that one to two histidine residues are involved in both the activation and inactivation processes. Difference spectra recorded during the reaction of NBA with DHPase and model compounds suggest that tryptophan residues play a role in the inactivation process, but this is not confirmed by amino acid analysis. The pH dependence of NBA modification shows pK[subscript a] value of 5.6 for the activation process and 6.0 for the inactivation process. |
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