1H-n.m.r. studies of the isolated activation segment from pig procarboxypeptidase A.
The isolated activation segment (asA) from pig pancreatic procarboxypeptidase A was studied by 1H-n.m.r. spectroscopy over a wide range of solution conditions. Isolated asA shows many characteristics of compactly folded globular proteins, such as the observation of perturbed positions for resonances from methyl groups, alpha-carbon atoms, histidine residues and the tyrosine residue. The single tyrosine residue (Tyr-70) exhibits a very high pKa, and both histidine and tyrosine residues show slow chemical modification (deuteration and iodination). In contrast, asA shows rapid NH exchange. Analysis of the spectra by pH titration and nuclear Overhauser effects revealed several residue interactions. Quantitative analysis of deuterium and tritium exchange allowed the assignment of the histidine C-2-H resonances to their respective residues in the sequence. His-66, the closest to the sites of proteolytic attack in the proenzyme, is shown to be the most accessible to solvent in procarboxypeptidase A. It was also shown that asA is thermally very stable ['melting' temperature (Tm) 88 degrees C] and requires a high urea concentration for denaturation (6.25 M, at pH 7.5). Evidence is presented for some degree of conformational flexibility in the premelting range, a feature that could be ascribed to the preponderance of helical secondary structure and to the lack of disulphide bridges. The free solution structure of asA is probably unchanged when it binds to carboxypeptidase A.
Vendrell
J
J
Departament de Bioquímica i Biologia Molecular (Facultat de Ciències), Universitat Autònoma de Barcelona, Bellaterra, Spain.
Avilés
F X
FX
Vilanova
M
M
Turner
C H
CH
Crane-Robinson
C
C
D000818:Animals; D002268:Carboxypeptidases; D043422:Carboxypeptidases A; D055598:Chemical Phenomena; D002621:Chemistry; D003903:Deuterium; D004789:Enzyme Activation; D004792:Enzyme Precursors; D006639:Histidine; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007455:Iodine; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D009994:Osmolar Concentration; D010179:Pancreas / Q000201:enzymology*; D010446:Peptide Fragments / Q000302:isolation & purification; D011487:Protein Conformation; D011489:Protein Denaturation; D013552:Swine; D014316:Tritium; D014443:Tyrosine; D014508:Urea
D004792:Enzyme Precursors; D010446:Peptide Fragments; D014316:Tritium; D014443:Tyrosine; D006639:Histidine; D014508:Urea; D007455:Iodine; D003903:Deuterium; D002268:Carboxypeptidases; D043422:Carboxypeptidases A