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The enzyme MetX is a homoserine <i>O</i>-acetyltransferase that catalyzes the first step in methionine biosynthesis and is essential for survival and virulence of various pathogens. It is an attractive target for antifungal and antibacterial drug development. MetX catalyzes the acetyl transfer from acetyl-CoA (AcCoA) to homoserine via a ping-pong mechanism involving an acyl-enzyme intermediate. The active site contains a Ser-His-Asp catalytic triad, which constitutes its core catalytic machinery. Here we investigated the mechanistic details of MetX from <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>MetX) using a combination of quantum mechanics/molecular mechanics (QM/MM) calculations, mutagenesis, and mass spectrometry. QM/MM calculations suggest that D320 of the catalytic triad participates in the proton transfer during homoserine acetylation, but not during acyl-enzyme formation. Experiments showed that a D320N substitution, which removes the proton-accepting capability of D320 as well as the p<i>K</i><sub>a</sub> modulation of H350 by D320, still allowed acyl-enzyme formation at a markedly reduced rate, but significantly impaired the production of acetyl-homoserine. To isolate the effect of D320's participation in proton transfer from its p<i>K</i><sub>a</sub> modulation role, we used QM/MM calculations to simulate a system where D320 could modulate H350 p<i>K</i><sub>a</sub> but not accept a proton. These calculations suggest that while D320's proton-accepting role is not required for the AcCoA reaction, it contributes thermodynamically in the homoserine reaction by lowering the energy of the forward pathway. Elucidating the mechanistic details of <i>Mtb</i>MetX reactions offers valuable insights that will facilitate the development of mechanism-based inhibitors, contributing to future therapeutic strategies.