The isomerization of beta-glucose-1-phosphate (beta G1P) to beta-glucose-6-phosphate (G6P) catalyzed by beta-phosphoglucomutase (beta PGM) has been examined using steady- and presteady-state kinetic analysis. In the presence of low concentrations of beta-glucose-1,6-bisphosphate (beta G16BP), the reaction proceeds through a Ping Pong Bi Bi mechanism with substrate inhibition (k(cat) = 65 s(-1), K-beta G1P = 15 mu M, K-beta G16BP = 0.7 uM, K-i = 122 mu M). If alpha G16BP is used as a cofactor, more complex kinetic behavior is observed, but the nonlinear progress curves can be fit to reveal further catalytic parameters (k(cat) = 74 s(-1), K-beta G1P = 15 mu M, K-beta G168P = 0.8 mu M, K-i = 122 mu M, K-alpha G16BP = 91 mu M for productive binding, K-alpha G16BP = 21 mu M for unproductive binding). These data reveal that variations in the substrate structure affect transition-state affinity (approximately 140 000-fold in terms of rate acceleration) substantially more than ground-state binding (110-fold in terms of binding affinity). When fluoride and magnesium ions are present, time-dependent inhibition of the beta PGM is observed. The concentration dependence of the parameters obtained from fitting these progress curves shows that a beta G1P center dot MgF3-center dot beta PGM inhibitory complex is formed under the reaction conditions. The overall stability constant for this complex is approximately 2 x 10(-16) M-5 and suggests an affinity of the MgF3- moiety to this transition-state analogue (TSA) of <= 70 nM. The detailed kinetic analysis shows how a special type of TSA that does not exist in solution is assembled in the active site of an enzyme. Further experiments show that under the conditions of previous structural studies, phosphorylated glucose only persists when bound to the enzyme as the TSA. The preference for TSA formation when fluoride is present, and the hydrolysis of substrates when it is not, rules out the formation of a stable pentavalent phosphorane intermediate in the active site of beta PGM.