The diabetogenic antibiotic streptozotocin modifies the tryptic digest pattern for peptides of the enzyme O-GlcNAc-selective N-acetyl-beta-d-glucosaminidase that contain amino acid residues essential for enzymatic activity.

Streptozotocin (STZ) inhibits O-GlcNAc-selective N-acetyl-beta-d-glucosaminidase (O-GlcNAcase), the enzyme that removes O-GlcNAc from proteins. The active site of the enzyme was recently proposed to include aspartates 174, 175, and 177, with STZ inhibition via a transition state analog. We explored the effect of STZ on the tryptic peptide digest pattern of O-GlcNAcase. LC/MS/MS analysis demonstrated that STZ modified two areas of the enzyme. One peptide, LGCFEIAK (894-901), in a C-terminal region previously proposed to possess O-GlcNAcase activity, was methylated by STZ. Another peptide, EYEIEFIYIASPGLDITFSNPK (128-149), was detected only after treatment with STZ and was in an N-terminal region, overlapping a glutamate-rich area containing an adjacent phenylalanine residue. No covalent modification of this peptide could be demonstrated. Detection of this peptide after treatment with STZ was accompanied by the simultaneous inability to detect the nearby peptide KLDQVSQFGCR (157-167), which contains a cysteine residue recently shown to be essential for enzymatic activity. To determine which of the first two peptides might also be important for O-GlcNAcase activity, site-specific mutagenesis was performed. Mutation of the N-terminal phenylalanine and serine residues resulted in almost complete inhibition of activity. In contrast, mutation of conserved C-terminal glycine and cysteine residues caused little inhibition of enzymatic activity. Together, these data extend the region of the active site N-terminally and give independent evidence to support the idea that STZ inhibits O-GlcNAcase through formation of a transition state analog that resides in the active site of the enzyme and in doing so alters its conformation and ensuing tryptic digest pattern.