Microtubules are cytoskeletal filaments with critical roles in cell division, cell motility, intracellular trafficking, and cilium function. In cells, subsets of microtubules are selectively marked by posttranslational modifications (PTMs), which control the ability of microtubule-associated proteins (MAPs) and molecular motors to engage microtubules. Detyrosination (ΔY) and ΔC2 are PTMs of α-tubulin, wherein one or two residues, respectively, are enzymatically removed from the C-terminus of the protein. How specific patterns of PTMs are generated in cells is not understood. Here, we use in vitro reconstitution assays to investigate the microtubule binding behavior of metallopeptidase MATCAP1 and the mechanism by which it generates ΔY and ΔC2 modifications of α-tubulin. We demonstrate that MATCAP1 preferentially binds to microtubules composed of tubulin subunits in an expanded conformation, which can be induced by preventing β-tubulin GTP hydrolysis, Taxol treatment, or kinesin-1 stepping. MATCAP1 binds to expanded microtubule lattices with long dwell time and sequentially removes the terminal tyrosine residue to generate ΔY-microtubules and the penultimate glutamate residue to generate ΔC2-microtubules. Thus, the lattice conformation of microtubules is a key factor that gates the binding and activity of MATCAP1.