THE CYSTEINES OF CATALASE HPII OF ESCHERICHIA-COLI, INCLUDING CYS438 WHICH IS BLOCKED, DO NOT HAVE A CATALYTIC ROLE

Site-directed mutagenesis of the katE gene of Escherichia coli was use d to change, individually and in combination, Cys438 and Cys669 to ser ine in catalase HPII. The Cys438-->Ser mutation caused a 30% reduction in the specific activity of the enzyme, whereas the Cys669-->Ser muta tion did not affect enzyme activity. The titration of free sulfhydryl groups in HPII revealed that Cys669 was reactive whereas Cys438 was un reactive. Properties of the modification on Cys438 included alkali lab ility, insensitivity to methylamine, hydroxylamine or reducing agents, and a mass determined by mass spectrometry to be approximately 43 +/- 2 Da. A hemithioacetal structure is consistent with these properties. Although free sulfhydryl groups do not play a significant role in the stability or catalytic mechanism of HPII, the sulfhydryl agent 2-merc aptoethanol caused a 50% inactivation of HPII along with an irreversib le change in the absorption spectrum of the protein. Other sulfhydryl agents, including dithiothreitol, cysteine and glutathione, and the or ganic peroxide, t-butylhydroperoxide, which cannot directly access the active site, do not affect HPII activity, but they do cause a small r eversible change in the absorption spectrum, possibly by a mechanism i nvolving superoxide.