The stereochemistry of the proton transfer in the reaction of phosphoenolbutyrate with enzyme I has been established. During the reaction of the pure Z isomer of this analogue of phosphoenolpyruvate with enzyme I, to yield phosphoenzyme I and 2-oxobutyrate, the substrate is protonated at C-3 from the 2re,3si face. This stereospecificity was established for the transfer of a proton to (Z)-phospho[3-D]enolbutyrate and for the transfer of a deuteron to (Z)-phospho[3-H]enolbutyrate. The E isomer of phosphoenolbutyrate is not a substrate for enzyme I. Accordingly, the reaction of phosphoenzyme I with 2-oxobutyrate yields exclusively the Z isomer of phosphoenolbutyrate, and only the pro-S proton at C-3 of 2-oxobutyrate is abstracted. A kinetic H/D isotope effect of 6.8 in this reaction demonstrates the rate-limiting nature of the proton-transfer step. The stereochemical analysis of 2-oxo[3(R)-H,D]butyrate and of 2-oxo-[3(S)-H,D]butyrate was carried out by using the pyruvate kinase catalyzed enolization of this compound. This enzymatic enolization, with phosphate as a cofactor, is rapid at neutral pH and is a highly stereospecific reaction: only the pro-R proton at C-3 of 2-oxobutyrate is exchanged with solvent. This reaction was also used to generate the pure 3R and 3S enantiomers of 2-oxo[3-H,D]butyrate. The degree of protonation/deuteration at C-3 of 2-oxobutyrate was detected from the fine structure of the methyl proton nuclear magnetic resonance signal.