Modification of the ( [formula omitted])-ATPase with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and with diethylpyrocarbonate. Effect on the conformational transition

Abstract

Modification of carboxyl groups of the ( Na + + K + )-ATPase with the water soluble 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide or of amino groups, histidine or alpha-amino groups, with diethylpyrocarbonate gives a pronounced decrease in apparent affinity for K + for transition from the Na +-form to the K +-form at a given Na + concentration. The fluorescence of eosin is used to monitor the conformational transitions. The decrease in apparent affinity for K + is due to an increase in the rate of the conformational transition from E 2 with K + occluded, E 2( K m +) , to E 2 with K + non-occluded, E 2 K m + , and to a lesser degree to a decrease in the rate of the reverse reaction, while there is little or no effect on the transition between E 2 in the absence of K + and E 1 Na n + . The conformational transition between E 2( K m +) and E 2 K m + is thus much more sensitive to a change in the tertiary and/or quaternary structure than the conformational transition between E 2 and E 1 Na n + . The pK values for the carboxyl groups are lower with the enzyme in the K +-form than in the Na +-form; in the Na +-form it seems to be lower than the lowest tested, pH 5.5. The diethylpyrocarbonate reactive amino group has a higher pK with the enzyme in the K +-form, about 8.0, than in the Na +-form, about 7.5. Plots of log K 0.5 for K + for reversal of the effect of Na + on the conformation vs. log[Na +] at a given pH, and vs. pH (from 6.8 to 8.0) at a given Na + concentration give straight lines, and the slope suggests that 4 Na + on E 1, E 1Na 4 +, is replaced by 2 K + + 1 H + on E 2, E 2(K 2 +)H +. Modification of the amino groups with diethylpyrocarbonate shifts the log K + vs. pH plot towards a higher K + value at a given pH, but the slope is as for control enzyme, while the log K + vs. log[Na +] plot becomes non-linear. None of the plots are linear after modification of the carboxyl groups with the carbodiimide.