The interaction of 4-fluoro-substituted benzeneboronic acids with subtilisin Carlsberg has been studied by F-19 NMR. At a field strength of 8.5 T, the inhibitors 4-fluoro- and 3-chloro-4-fluorobenzeneboronic acids are in slow exchange with the enzyme and exhibit bound shifts typical of boronate species. Dissociation rate constants determined by magnetization transfer were found to be pH independent in the range from 6.4 to 7.4, with values of 156 s-1 (4-fluoro) and 24 s-1 (3-chloro-4-fluoro) at pH 7.4, 21-degrees-C. These values are similar to previous temperature-jump determinations of the slow component of the dissociation of analogous ligands from subtilisin BPN’, demonstrating that the previously observed rates correspond to the boronate --> boronic acid transition. The interaction of 4-fluorobenzeneboronic acid (FBA) with 2-pyridylcarbinol, a model ligand for the histidine and serine groups of the active site, was also studied and demonstrated to involve chelation with both the hydroxyl oxygen and pyridyl nitrogen. Nitrogen-14 NMR studies indicate that, when complexed to the boronate, the pyridyl nitrogen exhibits a shift intermediate between the values for the protonated and unprotonated forms. This result is in close agreement with N-15 NMR studies of boronate inhibitors bound to the active site of [epsilon-N-15]histidine-labeled alpha-lytic protease. The dissociation rate constant for the 4-fluorobenzeneboronate-pyridylcarbinol complex of 22.5 s-1 is considerably slower than the dissociation rate constant for the corresponding subtilisin-boronate complex. The slower dissociation kinetics for the model complex is consistent with the conclusion that such bidentate chelation is not present for the boronate-subtilisin system under study.