1GHD: Crystal Structure Of The Glutaryl-7-aminocephalosporanic Acid Acylase By Mad Phasing

Glutaryl 7-aminocephalosporanic acid acylase of Pseudomonas sp. 130 (C130) was irreversibly inhibited in a time-dependent manner by two substrate analogs bearing side chains of variable length, namely 7beta-bromoacetyl aminocephalosporanic acid (BA-7-ACA) and 7beta-3-bromopropionyl aminocephalosporanic acid (BP-7-ACA). The inhibition of the enzyme with BA-7-ACA was attributable to reaction with a single amino acid residue within the beta-subunit proven by comparative matrix assisted laser desorption/ionization-time of flight mass spectrometry. Further mass spectrometric analysis demonstrated that the fourth tryptophan residue of the beta-subunit, Trp-B4, was alkylated by BA-7-ACA. By (1)H-(13)C HSQC spectroscopy of C130 labeled by BA-2-(13)C-7-ACA, it was shown that tryptophan residue(s) in the enzyme was alkylated, forming a carbon-carbon bond. Replacing Trp-B4 with other amino acid residues caused increases in K(m), decreases in k(cat), and instability of enzyme activity. None of the mutant enzymes except W-B4Y could be affinity-alkylated, but all were competitively inhibited by BA-7-ACA. Kinetic studies revealed that both BA-7-ACA and BP-7-ACA could specifically alkylate Trp-B4 of C130 as well as Tyr-B4 of the mutant W-B4Y. Because these alkylations were energy-requiring under physiological conditions, it is likely that the affinity labeling reactions were catalyzed by the C130 enzyme itself. The Trp-B4 residue is located in the middle of a characteristic alphabetabetaalpha sandwich structure. Therefore, a large conformational alteration during inhibitor binding and transition state formation is likely and suggests that a major conformational change is induced by substrate binding during the course of catalysis.
PDB ID: 1GHDDownload
MMDB ID: 23214
PDB Deposition Date: 2000/12/7
Updated in MMDB: 2017/10
Experimental Method:
x-ray diffraction
Resolution: 2.4  Å
Source Organism:
Similar Structures:
Biological Unit for 1GHD: dimeric; determined by author and by software (PISA)
Molecular Components in 1GHD
Label Count Molecule
Proteins (2 molecules)
Glutaryl-7-aminocephalosporanic Acid Acylase
Molecule annotation
Glutaryl-7-aminocephalosporanic Acid Acylase
Molecule annotation
* Click molecule labels to explore molecular sequence information.

Citing MMDB