THE AMINO ACID SEQUENCE OF THE TYROSINE-SENSITIVE 3-DEOXY-D-ARABINO - HEPTULOSONATE 7-PHOSPHATE SYNTHASE FROM ESCHERICHIA COLI K12
Abstract
The tyrosine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (EC 4.1.2.15) was sequenced. The enzyme was first purified from HE102/pKB45 by a rapid procedure that allows better than 50% recovery of the initial enzyme activity. Fragments necessary for protein sequencing were generated by digestion of the protein with trypsin, cyanogen bromide, Staphylococcus aureus V8 protease, and by partial acid digestion and were separated by a combination of fractionations on Sephadex and HPLC. The tryp+ophan-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (EC 4.1.2.15) was purified from 37-1/pJS101 and partially sequenced from fragments generated by cyanogen bromide digestion. This isoenzyme was judged to be a dimer with 41,000 dalton subunits by SDS polyacrylamide gel electrophoresis and gel filtration. The amino acid composition of the tyrosine- and tryptophan-sensitive isoenzymes were determined and compared to that of DAHP synthase (PHE). The amino terminal sequences of the three isoenzymes were then compared and a much more extensive sequence comparison between DAHP synthase (TYR) and DAHP synthase (TRP) is presented. The amino terminus of DAHP synthase (TYR) is also compared to hemoerythrin from the sea worm family Sipunculida. Two appendices describe the isolation of E. coli strains carrying a tyrosine-resistant form of DAHP synthase (TYR) and experiments leading to the labeling of the active site of this isoenzyme. These data should allow a rapid determination of the regions of the proteins involved in the enzymatic reaction and feedback inhibition of the enzyme. An appendix also describes experiments shedding some light on the overproduction of DAHP synthase (TYR) in E. coli strain HE102. These data suggest that a trans-acting element is present in strain HE102 that results not only in overproduction of DAHP synthase (TYR) but also in an inability of the cell to repress the synthesis of this enzyme.
Degree
Ph.D.
Subject Area
Biochemistry
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