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Immunological evidence for substrate-induced conformational alterations in human carbonic anhydrase B.
The inhibition by specific antibody of carbonic anhydrase B activity towards bicarbonate was uncompetitive (Ki 9.5 X 10(-7) M) whereas that of activity towards p-nitrophenylacetate was mixed-type (Ki 9.2 X 10(-7) M). Differences in the immunological reactivity of the enzyme in the presence of the substrates was documented by quantitative precipitin tests and by the Farr technique. In the presence of bicarbonate, nearly half of the antigenic determinants of the enzyme were altered to such an extent that antibodies directed against these determinants in the native form did not bind. This antibody fraction was separated and shown to react with the native enzyme in different molar ratios than the total antibody population. It produced only a very limited inhibition of the activity towards CO2 and p-nitrophenyl acetate. The immunological reactivity of the enzyme with both the total antibody population and the fraction that is nonreactive in the presence of bicarbonate was identical in the absence of substrates and in the presence of CO2 and p-nitrophenyl acetate. It is suggested that the native enzyme form exhibits enzymatic activity towards CO2 and p-nitrophenyl acetate, whereas in the presence of bicarbonate a "conformational adaptation" is induced.
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