Identification of active site residues of chorismate mutase-prephenate dehydrogenase from Escherichia coli

Biochemistry
D Christendat, J Turnbull

Abstract

Chemical modification studies of the bifunctional enzyme chorismate mutase-prephenate dehydrogenase and mass spectral analysis of peptide fragments containing modified residues are presented. The reaction with diethyl pyrocarbonate (DEPC) results in the modification of several enzymic groups, including a single histidine group essential for dehydrogenase activity and a single lysine residue essential for mutase activity. This conclusion is based on the following evidence. (1) Hydroxylamine rapidly restores dehydrogenase activity to the DEPC-inactivated enzyme without restoring mutase activity. (2) Mutase activity is also lost upon treatment of the enzyme with trinitrobenzene sulfonate. (3) The reactivity of the dehydrogenase to DEPC increases with pH, suggesting the participation of a group with a pKa of 7.0 in the dehydrogenase reaction. (4) Two peptides identified by differential peptide mapping had mass values matching those calculated for peptides comprising residues 127-135 (containing His131) and residues 36-48 (containing Lys37). In support of the idea that the residues being modified are within the active sites, we show that the substrates prephenate and nicotinamide adenine dinucleotide (NAD+) offer protection against ...Continue Reading

References

Jan 1, 1970·Annual Review of Biochemistry·A N Glazer

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Citations

Oct 13, 2001·Bioorganic & Medicinal Chemistry Letters·M R BirckR W Woodard
Mar 11, 1998·Natural Product Reports·P M Dewick
Jul 13, 2011·Experimental Diabetes Research·Haohua Qian, Harris Ripps
May 30, 2006·Protein Science : a Publication of the Protein Society·Julie BonvinJoanne L Turnbull

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