Chemical modification of catalytic site of lipase from wheat germ: altered structure-activity profile.
Wheat germ lipase (WGL) was inactivated by chemical modification of histidine, serine and carboxyl groups of Asp/Glu residues with diethyl pyrocarbonate (DEPC), phenyl methyl sulfonyl fluoride (PMSF) and 1-ethyl-3-(3-dimethylaminopropyl) carbodi-imide (EDC), respectively. Loss of activity of WGL was concentration dependent of the inhibitor and at 30 mM PMSF most of the activity of the enzyme was lost. The stoichiometry of modification showed one mole of histidine, serine and two moles of carboxyl groups modified per mole of protein. Kinetic measurements indicated that the inhibition of the enzyme was competitive in nature. The modified enzyme was further characterized by far UV-circular dichroic measurements of the secondary structure and fluorescence spectroscopy. PMSF-modified enzyme showed decreased thermal stability, whereas no change was observed in DEPC-modified enzyme as evidenced by differential scanning calorimetry. These studies indicate that histidine, serine and Asp/Glu residues play an important role in the catalytic function of WGL. The mechanism of loss of activity is due to minor conformational change in the microenvironment of the active site rather than the gross conformational change of the molecule itself.
Department of Protein Chemistry and Technology, Central Food Technological Research Institute, Mysore 570 013, India.
K N
KN
Gopalakrishna
P Ramesh
PR
Kumar
V
V
Prakash
D001224:Aspartic Acid / Q000737:chemistry; D002152:Calorimetry, Differential Scanning / Q000379:methods; D020134:Catalytic Domain; D002942:Circular Dichroism; D004305:Dose-Response Relationship, Drug; D006639:Histidine / Q000737:chemistry; D006358:Hot Temperature; D007700:Kinetics; D008049:Lipase / Q000737:chemistry*; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D012694:Serine / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000379:methods; D013056:Spectrophotometry, Ultraviolet / Q000379:methods; D013329:Structure-Activity Relationship; D013696:Temperature; D014908:Triticum / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*
D001224:Aspartic Acid; D012694:Serine; D006639:Histidine; D008049:Lipase