ISSN:
1573-4919
Source:
Springer Online Journal Archives 1860-2000
Topics:
Biology
,
Chemistry and Pharmacology
,
Medicine
Notes:
Summary δ-Chymotrypsin has previously been reported to undergo a slow isomerization between a high pH form and a neutral pH form (pK = 9) as observed by its reactivity towards a variety of substrates. [Garel, J-R and Labouesse, B. (1973) Eur. J. Biochem., 39, 293–300, Fersht, A. R. (1972) J. Mol. Biol. 64, 497–509] Since slow transitions, e.g., isomerizations, between two or more enzyme forms which have different kinetic properties can produce kinetic cooperativity for either monomeric or oligomeric enzymes [Ainslie, G. R., Jr., Shill, J. P. and Neet, K. E. (1972) J. Biol. Chem. 247, 7088–7096], we have investigated whether δ-chymotrypsin might show cooperativity with some substrates. Careful statistical analysis of the steady state kinetics has demonstrated that negative cooperativity with the substrate N-CBZ-l-tryptophan p-nitrophenyl ester occurs at pH 9 and 25 °C, with a Hill coefficient of about 0.6. Mechanisms which involve interactions between catalytic sites cannot be the source of the cooperativity of δ-chymotrypsin since it is monomeric and has only a single catalytic site. For these reasons the δ-chymotrypsin kinetics presented here have been interpreted in terms of the slow transition mechanism for kinetic cooperativity. 1. The negative cooperativity with the tryptophan substrate disappears if the temperature is decreased or the pH is lowered. This observation is in accord with the slow transition mechanism since the fraction of δ-chymotrypsin present in the high pH form is kinetically insignificant under these conditions. 2. Alterations in the nature of the substrate and ionic strength also eliminate the observation of negative cooperativity. Such a loss in cooperativity is consistent with the slow transition mechanism in which the cooperativity may be sensitive to small changes in kinetic constants.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/BF00220737
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