1cia
From Proteopedia
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REPLACEMENT OF CATALYTIC HISTIDINE-195 OF CHLORAMPHENICOL ACETYLTRANSFERASE: EVIDENCE FOR A GENERAL BASE ROLE FOR GLUTAMATE
Overview
The imidazole N epsilon 2 of His-195 plays an essential part in the, proposed general base mechanism of chloramphenicol acetyltransferase, (CAT), hydrogen bonding to and a abstracting a proton from the primary, hydroxyl group of chloramphenicol. Replacement of His-195 by alanine or, glutamine results in apparent decreases in kcat of (9 x 10(5)- and (3 x, 10(5))-fold, respectively, whereas Km values for both substrates, (chloramphenicol and acetyl-CoA) are similar to those of wild-type CAT., The structure of Gln-195 CAT has been solved at 2.5-A resolution and is, largely isosteric with that of wild-type CAT. Substitution of His-195 by, glutamate resulted in a (5 x 10(4))-fold decrease in kcat together with a, 3-fold increase in the Km for chloramphenicol. Direct determination of, binding constants for both substrates demonstrated that these, substitutions result in only small decreases in the affinity of CAT for, acetyl-CoA (Kd values increased 2- to 3-fold), whereas chloramphenicol Kd, values are elevated 26-, 20-, and 53-fold for Ala-195 CAT, Gln-195 CAT and, Glu-195 CAT, respectively. The pH dependence of kcat/Km yields apparent, pKa values of 6.5 and 6.7 for Ala-195 CAT and Gln-195 CAT, respectively, which are very similar to that (6.6) determined for the ionization of, His-195 in wild-type CAT. In contrast, the pH dependence of kcat/Km for, Glu-195 CAT (pKa = 8.3) is very different from that of wild-type, CAT.(ABSTRACT TRUNCATED AT 250 WORDS)
About this Structure
1CIA is a Single protein structure of sequence from Escherichia coli with CO and BME as ligands. Active as Chloramphenicol O-acetyltransferase, with EC number 2.3.1.28 Full crystallographic information is available from OCA.
Reference
Replacement of catalytic histidine-195 of chloramphenicol acetyltransferase: evidence for a general base role for glutamate., Lewendon A, Murray IA, Shaw WV, Gibbs MR, Leslie AG, Biochemistry. 1994 Feb 22;33(7):1944-50. PMID:7906544
Page seeded by OCA on Tue Nov 20 12:28:48 2007
