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.