Studies on the chymotrypsin-catalysed hydrolysis of resorufin acetate and resorufin bromoacetate.
Resorufin bromoacetate is a substrate that is rapidly hydrolysed by chymotrypsin. The reaction shows a pre-steady-state burst phase that may be observed by stopped flow spectrophotometry if precautions are taken against spontaneous hydrolysis of the substrate. The strongly activating effect that the presence of the bromine atom has on the adjacent carbonyl group is reflected in the relative sizes of the kcat values for resorufin bromoacetate and resorufin acetate (e.g., 740 to 1, at pH 6) and the burst rate constants (e.g., 350 to 1, at pH 7 using 0.1 mM substrate). The pH-dependence of kcat for both substrates shows the involvement of an enzymic group of pKa about 7. With resorufin bromoacetate, a burst and a steady-state rate are still observable at pH 3.0. Unlike the case with aldehyde dehydrogenase, resorufin bromoacetate does not act as an inactivator of chymotrypsin and there is little or no incorporation of covalently-linked label when chymotrypsin and resorufin bromoacetate are incubated together. The different modes of behaviour of the two enzymes are attributable to the 'hard' or 'soft' character of the attacking enzymic nucleophilic groups.
Kitson
T M
TM
Institute of Fundamental Sciences: Chemistry, Massey University, Palmerston North, New Zealand. t.m.kitson@massey.ac.nz
D002918:Chymotrypsin / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D010078:Oxazines / Q000378:metabolism* / Q000494:pharmacology; D011485:Protein Binding; D013056:Spectrophotometry, Ultraviolet
D010078:Oxazines; C113486:resorufin bromoacetate; C105548:resorufin acetate; D002918:Chymotrypsin