A MASS SPECTROMETRY-BASED APPROACH FOR PROBING ENZYME ACTIVE-SITES - IDENTIFICATION OF GLU-127 IN CELLULOMONAS-FIMI EXOGLYCANASE AS THE RESIDUE MODIFIED BY N-BROMOACETYL CELLOBIOSYLAMINE

We have identified the residue in Cellulomonas fimi exoglycanase modif ied by N-bromoacetyl cellobiosylamine as Glu 127 using a new combinati on of experimental approaches. The enzyme was quantitatively inhibited with the affinity label N-bromoacetyl cellobiosylamine and cleaved wi th pepsin. The N-acetyl cellobiosylamine-modified peptide was identifi ed by comparative peptide mapping of the digests derived from labeled and unlabeled proteins by reverse-phase high-performance liquid chroma tography connected on-line to an electrospray ionization mass spectrom eter. The modified residue in the labeled peptide was determined by us ing a novel protein sequencing chemistry which is based on monitoring the amino acid derivatives released by stepwise peptide degradation us ing electrospray ionization mass spectrometry. Tandem mass spectrometr y was used for further structural characterization of the cleaved resi due. We show that the residue modified by N-bromoacetyl cellobiosylami ne is Glu 127. This residue has been identified previously as the acid -base catalyst by using a combination of mutagenic and kinetic analyse s. Our results therefore demonstrate the usefulness of this type of af finity label in identifying important catalytic residues in glycosidas es and suggest that this new experimental approach can be applied gene rally to any labeled protein in which the mass of the label is known a nd thus represents an alternative approach to the current methods used to identify labeled residues within proteins. (C) 1996 Academic Press , Inc.