Cleavage of pyridoxal kinase into two structural domains: kinetics of proteolysis monitored by emission anisotropy.
Two sulfhydryl residues/dimer of pyridoxal kinase react with iodoacetamide fluorescence (IAF) to yield catalytically active species. Limited chymotryptic digestion of IAF pyridoxal kinase resulted in the release of two fragments of 24 and 16 KDA. One of the fragments (16 KDA) is labeled with IAF. After complete tryptic digestion of IAF-pyridoxal kinase, only one peptide labeled with IAF was separated by reverse-phase HPLC and its amino acid sequence determined by automated Edman degradation. The kinetics of chymotryptic cleavage of IAF-pyridoxal kinase was monitored by steady-state emission anisotropy measurements. Analysis of the kinetic results revealed that the rate of proteolysis is significantly reduced by the substrate pyridoxal (0.2 mM). ATP (1 mM) does not influence the rate of proteolysis. The technique of emission anisotropy was also applied to monitor the effect of viscosity on the rate of proteolysis. A kinetic model is proposed to explain the mechanism of limited proteolysis. The model is based on the assumption that unfolding of the native conformation of the protein-substrate complex plays a dominant role in proteolysis.
Churchich
J E
JE
Department of Biochemistry, University of Tennessee, Knoxville 37996-8040.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002918:Chymotrypsin; D004591:Electrophoresis, Polyacrylamide Gel; D005452:Fluoresceins; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D011731:Pyridoxal Kinase / Q000737:chemistry*; D012756:Sheep; D013056:Spectrophotometry, Ultraviolet; D013057:Spectrum Analysis / Q000379:methods; D013438:Sulfhydryl Compounds / Q000032:analysis; D014357:Trypsin
D000596:Amino Acids; D005452:Fluoresceins; D010446:Peptide Fragments; D013438:Sulfhydryl Compounds; C015296:5-iodoacetamidofluorescein; D011731:Pyridoxal Kinase; D002918:Chymotrypsin; D014357:Trypsin