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CellularO-linkedN-acetylglucosamine (O-GlcNAc) levels are modulated by two enzymes: uridine diphosphate-N-acetyl-Dglucosamine: polypeptidyltransferase (OGT) and O-GlcNAcase (OGA). To quantitatively address the activity of these enzymes on protein substrates, we generated five structurally diverse proteins in both unmodified and O-GlcNAc-modified states. We found a remarkably invariant upper limit for kcat/Km values for human OGA (hOGA)-catalyzed processing of these modified proteins, which suggests that hOGA processing is driven by the GlcNAc moiety and is independent of the protein. Human OGT(hOGT) activity ranged more widely, by up to 15-fold, suggesting that hOGT is the senior partner in fine tuning protein O-GlcNAc levels. This was supported by the observation that Km,app values for UDP-GlcNAc varied considerably (from 1 μM to over 20 μM), depending on the protein substrate, suggesting that some OGT substrates will be nutrient-responsive, whereas others are constitutively modified. The ratios of kcat/Km values obtained fromhOGTandhOGAkinetic studies enable a prediction of the dynamic equilibrium position of O-GlcNAc levels that can be recapitulated in vitro and suggest the relative O-GlcNAc stoichiometries of target proteins in the absence of other factors. We show that changes in the specific activities of hOGTandhOGAmeasured in vitro on calcium/calmodulin-dependent kinase IV (CaMKIV) and its pseudophosphorylated form can account for previously reported changes in CaMKIV O-GlcNAc levels observed in cells. These studies provide kinetic evidence for the interplay between O-GlcNAc and phosphorylation on proteins and indicate that these effects can be mediated by changes in hOGT and hOGA kinetic activity. [ABSTRACT FROM AUTHOR]