Identification of catalytically essential amino acid residues of penicillin G acylase obtained from a mutant of Escherichia coli ATCC 11105


Kazan D., Erarslan A.

PROCESS BIOCHEMISTRY, cilt.36, ss.861-867, 2001 (SCI İndekslerine Giren Dergi) identifier identifier

  • Cilt numarası: 36
  • Basım Tarihi: 2001
  • Doi Numarası: 10.1016/s0032-9592(00)00283-1
  • Dergi Adı: PROCESS BIOCHEMISTRY
  • Sayfa Sayıları: ss.861-867

Özet

The chemical modification of penicillin G acylase (PGA) obtained from a mutant of Escherichia coli ATCC 11105 was studied in order to identify the catalytically essential amino acid residues of the enzyme. The modification of PGA by serine specific phenylmethylsulphonylfluoride (PMSF) and tryptophan specific N-bromosuccinimide (NBS) resulted in the complete inactivation of the enzyme. Modification by arginine specific phenylglyoxal and lysine specific ethylacetimidate caused the partial inactivation of the enzyme and partial protection of enzyme activity was observed when modifications were carried out in the presence of the strong competitive inhibitor penicillin G sulphoxide (PenGSO). Enzyme activity was not affected by the modification of cysteine specific ethylmaleiimide and histidine specific reagents tosyl-L-lysine-chloromethyl ketone (TLCK) and tosyl-L-phenylalanine-chloromethyl ketone (TPCK). The inactivation of PGA by amino acid specific modifying reagents obeyed first-order kinetics. The kinetic constants (V-m, K-m and k(cat) values) and pH-activity profiles of native and modified PGA preparations were also investigated. The results of these investigations showed that the active site of the enzyme contained a catalytically essential serine residue. It may be argued that the modification of arginine residues induces the conformational change of the entire enzyme molecule. These residues are located at a site other than the active site of the enzyme and are not essential for catalysis. The modification of lysine residues induces conformational changes in the active site of the enzyme but these residues can contribute neither catalysis nor substrate binding. (C) 2001 Elsevier Science Ltd. All rights reserved.