Site-directed mutagenesis of essential carboxylic residues in Clostridium thermocellum endoglucanase CelD.

12 carboxyl residues of the Clostridium thermocellum endoglucanase CelD were mutated to alanine. The specific activity of five of the mutated proteins was 1% or less that of wild type. The Ca2+ binding isotherms of these five were similar to those of wild type CelD, consistent with the fact that none of the mutated residues is observed to be directly involved in Ca2+ binding in the three-dimensional structure of the protein (Juy, M., Anit, A. G., Alzuri, P. M., Poljak, R. J., Claeyssens, M., Béguin, P., and Aubert, J.-P., manuscript in preparation) and suggesting that the mutations did not result in gross alterations of the tertiary structure. Analysis of the physico-chemical and enzymatic properties of the five purified mutated proteins and consideration of their position in the three-dimensional structure suggest that carboxyl groups identified may play roles as a general acid catalyst and a source of negative charge in stabilizing a carbonium ion intermediate. Among mutated residues, Glu-555 appears as the most likely candidate for participating in the catalytic mechanism of endoglucanase CelD.