1GM8

Crystal structures of penicillin acylase enzyme-substrate complexes: Structural insights into the catalytic mechanism

Summary for 1GM8

• Entry DOI: 10.2210/pdb1gm8/pdb
• Related: 1AI4 1AI5 1AI6 1AI7 1AJN 1AJP 1AJQ 1CP9 1E3A 1FXH 1FXV 1GK9 1GKF 1GM7 1GM9 1PNK 1PNL 1PNM
• Descriptor: PENICILLIN G ACYLASE ALPHA SUBUNIT, PENICILLIN G ACYLASE BETA SUBUNIT, CALCIUM ION, ... (5 entities in total)
• Functional Keywords: antibiotic resistance, hydrolase
• Biological source: ESCHERICHIA COLI; More
• Total number of polymer chains: 2
• Total formula weight: 86617.78

Authors

McVey, C.E.,Walsh, M.A.,Dodson, G.G.,Wilson, K.S.,Brannigan, J.A. (deposition date: 2001-09-11, release date: 2001-11-28, Last modification date: 2024-05-08)

Primary citation

Mcvey, C.E.,Walsh, M.A.,Dodson, G.G.,Wilson, K.S.,Brannigan, J.A.
Crystal Structures of Penicillin Acylase Enzyme- Substrate Complexes: Structural Insights Into the Catalytic Mechanism
J.Mol.Biol., 313:139-, 2001

PubMed Abstract: The crystal structure of penicillin G acylase from Escherichia coli has been determined to a resolution of 1.3 A from a crystal form grown in the presence of ethylene glycol. To study aspects of the substrate specificity and catalytic mechanism of this key biotechnological enzyme, mutants were made to generate inactive protein useful for producing enzyme-substrate complexes. Owing to the intimate association of enzyme activity and precursor processing in this protein family (the Ntn hydrolases), most attempts to alter active-site residues lead to processing defects. Mutation of the invariant residue Arg B263 results in the accumulation of a protein precursor form. However, the mutation of Asn B241, a residue implicated in stabilisation of the tetrahedral intermediate during catalysis, inactivates the enzyme but does not prevent autocatalytic processing or the ability to bind substrates. The crystal structure of the Asn B241 Ala oxyanion hole mutant enzyme has been determined in its native form and in complex with penicillin G and penicillin G sulphoxide. We show that Asn B241 has an important role in maintaining the active site geometry and in productive substrate binding, hence the structure of the mutant protein is a poor model for the Michaelis complex. For this reason, we subsequently solved the structure of the wild-type protein in complex with the slowly processed substrate penicillin G sulphoxide. Analysis of this structure suggests that the reaction mechanism proceeds via direct nucleophilic attack of Ser B1 on the scissile amide and not as previously proposed via a tightly H-bonded water molecule acting as a "virtual" base.

PubMed: 11601852
DOI: 10.1006/JMBI.2001.5043

Experimental method

X-RAY DIFFRACTION (2 Å)