Stereospecificity, substrate, and inhibitory properties of nucleoside diphosphate analogs for creatine and pyruvate kinases

Antiviral -P-borano substituted NTPs are promising chain terminators targeting HIV reverse transcriptase (RT). Activation of antiviral nucleoside diphosphates (NDPs) to NTPs may be carried out by pyruvate kinase (PK) and creatine kinase (CK). Herein, are presented the effects of nucleobase, ribose, and -phosphate substitutions on substrate specificities of CK and PK. Both enzymes showed two binding modes and negative cooperativity with respect to substrate binding. The stereospecificity and inhibition of ADP phosphorylation by -P-borano substituted NDP (NDPB) stereoisomers were also investigated. The Sp-ADPB isomer was a 70-fold better substrate for CK than the Rp isomer, whereas PK preferred the Rp isomer of NDPBs. For CK, the Sp-ADPB isomer was a competitive inhibitor; for PK, the Rp-ADPB isomer was a poor competitive inhibitor and the Sp-ADPB isomer was a poor non-competitive inhibitor. Taken together, these data suggest that, although the Rp-NDPB isomer would be minimally phosphorylated by CK or PK, it should not inhibit either enzyme.



ARTICLE

Creatine Kinase–Mediated ATP Supply Fuels Actin-Based Events in Phagocytosis

Phagocytosis requires locally coordinated cytoskeletal rearrangements driven by actin polymerization and myosin motor activity. How this actomyosin dynamics is dependent upon systems that provide access to ATP at phagosome microdomains has not been determined. We analyzed the role of brain-type creatine kinase (CK-B), an enzyme involved in high-energy phosphoryl transfer. We demonstrate that endogenous creatine kinase (CK-B), in macrophages is mobilized from the cytosolic pool and coaccumulates with F-actin at nascent phagosomes. Live cell imaging with XFP-tagged CK-B and β-actin revealed the transient and specific nature of this partitioning process. Overexpression of a catalytic dead CK-B or CK-specific cyclocreatine inhibition caused a significant reduction of actin accumulation in the phagocytic cup area, and reduced complement receptor–mediated, but not Fc-γR–mediated, ingestion capacity of macrophages. Finally, we found that inhibition of CK-B affected phagocytosis already at the stage of particle adhesion, most likely via effects on actin polymerization behavior. We propose that CK-B activity in macrophages contributes to complement-induced F-actin assembly events in early phagocytosis by providing local ATP supply.