ADP inhibits function of the ABC transporter cystic fibrosis transmembrane conductance regulator via its adenylate kinase activity

ADP interacts with the nucleotide-binding domains (NBDs) of the cystic fibrosis transmembrane conductance regulator (CFTR) to inhibit its Cl(-) channel activity. Because CFTR NBD2 has reversible adenylate kinase activity (ATP + AMP ⇆ ADP + ADP) that gates the channel, we asked whether ADP might inhibit current through this enzymatic activity. In adenylate kinases, binding of the two ADP molecules is cooperative. Consistent with this hypothesis, CFTR current inhibition showed positive cooperativity for ADP. We also found that ADP inhibition of current was attenuated when we prevented adenylate kinase activity with P(1),P(5)-di(adenosine-5′) pentaphosphate. Additional studies suggested that adenylate kinase-dependent inhibition involved phosphotransfer between two nucleotide diphosphates. These data indicate that the adenylate kinase reaction at NBD2 contributed to the inhibitory effect of ADP. Finding that ADP inhibits function via an adenylate kinase activity also helps explain the earlier observation that mutations that disrupt adenylate kinase activity also disrupt ADP inhibition. Thus, the results reveal a previously unrecognized mechanism by which ADP inhibits an ABC transporter

Intracellular Positioning of Isoforms Explains an Unusually Large Adenylate Kinase Gene Family in the Parasite Trypanosoma brucei.

Adenylate kinases occur classically as cytoplasmic and mitochondrial enzymes, but the expression of seven adenylate kinases in the flagellated protozoan parasite Trypanosoma brucei (order, Kinetoplastida; family, Trypanosomatidae) easily exceeds the number of isoforms previously observed within a single cell and raises questions as to their location and function. We show that a requirement to target adenylate kinase into glycosomes, which are unique kinetoplastid-specific microbodies of the peroxisome class in which many reactions of carbohydrate metabolism are compartmentalized, and two different flagellar structures as well as cytoplasm and mitochondrion explains the expansion of this gene family in trypanosomes. The three isoforms that are selectively built into either the flagellar axoneme or the extra-axonemal paraflagellar rod, which is essential for motility, all contain long N-terminal extensions. Biochemical analysis of the only short form trypanosome adenylate kinase revealed that this enzyme catalyzes phosphotransfer of -phosphate from ATP to AMP, CMP, and UMP acceptors; its high activity and specificity toward CMP is likely to reflect an adaptation to very low intracellular cytidine nucleotide pools. Analysis of some of the phosphotransfer network using RNA interference suggests considerable complexity within the homeostasis of cellular energetics. The anchoring of specific adenylate kinases within two distinct flagellar structures provides a paradigm for metabolic organization and efficiency in other flagellates