Multiple effects of chemical reagent on enzyme: o-phthalaldehyde-induced inactivation, dissociation and partial unfolding of lactate dehydrogenase from pig heart.
The effects of o-phthalaldehyde (OPTA) on lactate dehydrogenase (LDH) have been studied by following changes in enzymatic activity, aggregation state and conformation. Treatment with OPTA resulted in pseudo first-order inactivation of LDH over a wide concentration range of the inhibitor, and the second-order rate constant was estimated to be 1.52M(-1)s(-1). The loss of enzyme activity was concomitant with the increases in absorbance at 337nm and fluorescence intensity at 405nm. Complete loss of enzyme activity was accompanied by the formation of approximately 4mol isoindole derivatives per mole LDH subunit. Cross-linking experiments verified enzyme dissociation during OPTA modification, which could be attributed to the modification of both thiol groups and lysine residues. Circular dichroism (CD) spectra showed that the secondary structure of the OPTA-modified enzyme decreased correspondingly. Comparison of the inactivation with the conformational changes of the enzyme suggests that the active site of the enzyme exhibits greater conformational flexibility than the enzyme molecule as a whole. It is concluded that OPTA modification has multiple effects on LDH, including its inactivation, dissociation and partial unfolding.
Zheng
Yan-bin
YB
Department of Biological Science and Biotechnology, Tsinghua University, Beijing, China.
Wang
Zheng
Z
Chen
Bao-yu
BY
Wang
Xi-cheng
XC
D000818:Animals; D002942:Circular Dichroism; D003432:Cross-Linking Reagents / Q000737:chemistry; D005976:Glutaral / Q000737:chemistry; D006321:Heart; D007770:L-Lactate Dehydrogenase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013552:Swine; D009764:o-Phthalaldehyde / Q000737:chemistry / Q000494:pharmacology*
D003432:Cross-Linking Reagents; D009764:o-Phthalaldehyde; D007770:L-Lactate Dehydrogenase; D005976:Glutaral