Differences between CTP and ATP as substrates for the (Na + K)-ATPase

Abstract

CTP was a poorer substrate than ATP when substituted in the (Na + K)-ATPase reaction assay, not only in terms of K m but also of V. CDP was a poorer inhibitor than ADP, so product inhibition cannot account for CTP being a poorer substrate. In the Na-ATPase reaction, which the enzyme also catalyzes, substituting CTP for ATP resulted in greater activity, arguing against CTP being less effective than ATP in forming the enzyme-phosphate intermediate common to both reactions. Ligands that favor the E 2 conformational state of the enzyme, K +, Mg 2+, and Mn 2 +, inhibited the (Na + K)-CTPase reaction more than the (Na + K)-ATPase. Conversely, Triton X-100, which favors the E 1 conformational state of the enzyme, K +, Mg 2+, and Mn 2+, inhibited the (Na + K)-CTPase ATPase reaction but stimulated the (Na + K)-CTPase. Although the (Na + K)-ATPase reaction sequence probably involves cyclical interconversion between E 1 and E 2 conformational states (and is thus inhibitable by ligands favoring either state), the K-phosphatase reaction catalyzed by the enzyme apparently functions entirely in the E 2 state. This reaction is better stimulated by CTP plus Na + than by ATP plus Na +; moreover, CTP lessens inhibition by Triton X-100, and ATP lessens inhibition by inorganic phosphate (which reacts with the E 2 state). These observations indicate that CTP is a poorer substrate than ATP because it is less effective in promoting conversion of E 2 to E 1, essential for the (Na + K)-dependent reaction mechanism. However, contrary to this rationale, dimethyl sulfoxide stimulated the (Na + K)-CTPase reaction although by other criteria, including inhibition of the (Na + K)-ATPase, the reagent appears to favor the E 2 over the E 1 conformational state.