Trypsin-modified alkaline phosphatase. Formation of apoenzyme monomer and hybrid dimer.
The cleavage of an amino-terminal decapeptide from Escherichia coli alkaline phosphatase has been previously described (Roberts, C. H., and Chlebowski, J. F. (1984) J. Biol. Chem. 259, 729-733) by this laboratory. The modest reduction in specific activity of the modified enzyme is paralleled by an apparent alteration in the Zn(II) affinity at one of the three active center metal ion binding sites. In contrast to the behavior of the native enzyme, formation of the metal-free apoprotein results in an irreversible loss of catalytic activity; phosphohydrolase activity is not restored on addition of Zn(II) and Mg(II). Differential scanning calorimetry and velocity sedimentation data indicate that the apo form of the modified enzyme exists as a monomer form which, while capable of binding Zn(II) does not readily reassociate to active dimer. Processive cleavage of the amino termini of the dimer by trypsin results in the transient formation of a hybrid dimer consisting of cleaved and uncleaved subunits. This species can be directly observed and isolated by taking advantage of the differential chromatographic mobility of the native "isozymes" and the resulting products. Coupled with improved procedures for the preparation of the modified protein, these data indicate that the amino-terminal modification results in alterations in the subunit interface domain and provides a species (the hybrid dimer) for the investigation of the propagation of these effects.
D000469:Alkaline Phosphatase / Q000378:metabolism*; D001051:Apoenzymes / Q000494:pharmacology*; D001059:Apoproteins / Q000494:pharmacology*; D002152:Calorimetry, Differential Scanning; D002712:Chlorides; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D015636:Magnesium Chloride; D008970:Molecular Weight; D014357:Trypsin / Q000494:pharmacology*; D015032:Zinc / Q000494:pharmacology; D017967:Zinc Compounds
D001051:Apoenzymes; D001059:Apoproteins; D002712:Chlorides; D046911:Macromolecular Substances; D017967:Zinc Compounds; D015636:Magnesium Chloride; C016837:zinc chloride; D000469:Alkaline Phosphatase; D014357:Trypsin; D008274:Magnesium; D015032:Zinc