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 pK(a) 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 His 131) 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 inactivation of dehydrogenase activity while inactivation of mutase activity can be prevented by prephenate and a transition state analogue (3-endo-8-exo)-8-hydroxy-2-oxabicyclo[3.3.1]-non-6-ene-3, 5-dicarboxylic acid (endo-oxabicyclic diacid). Lys37 is conserved among several chorismate mutases and may participate in catalysis by interacting with an ether oxygen between C-5 and C-8 of chorismate in the transition state. His 131 may be assisting in a hydride transfer from prephenate to NAD(+) in the dehydrogenase reaction.
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AUSTRALIAN NATL UNIV, JOHN CURTIN SCH MED RES, DEPT BIOCHEM, CANBERRA, ACT 2601, AUSTRALIAAUSTRALIAN NATL UNIV, JOHN CURTIN SCH MED RES, DEPT BIOCHEM, CANBERRA, ACT 2601, AUSTRALIA
BHOSALE, SB
;
ROOD, JI
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AUSTRALIAN NATL UNIV, JOHN CURTIN SCH MED RES, DEPT BIOCHEM, CANBERRA, ACT 2601, AUSTRALIAAUSTRALIAN NATL UNIV, JOHN CURTIN SCH MED RES, DEPT BIOCHEM, CANBERRA, ACT 2601, AUSTRALIA
ROOD, JI
;
SNEDDON, MK
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AUSTRALIAN NATL UNIV, JOHN CURTIN SCH MED RES, DEPT BIOCHEM, CANBERRA, ACT 2601, AUSTRALIAAUSTRALIAN NATL UNIV, JOHN CURTIN SCH MED RES, DEPT BIOCHEM, CANBERRA, ACT 2601, AUSTRALIA
SNEDDON, MK
;
MORRISON, JF
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AUSTRALIAN NATL UNIV, JOHN CURTIN SCH MED RES, DEPT BIOCHEM, CANBERRA, ACT 2601, AUSTRALIAAUSTRALIAN NATL UNIV, JOHN CURTIN SCH MED RES, DEPT BIOCHEM, CANBERRA, ACT 2601, AUSTRALIA
机构:
AUSTRALIAN NATL UNIV, JOHN CURTIN SCH MED RES, DEPT BIOCHEM, CANBERRA, ACT 2601, AUSTRALIAAUSTRALIAN NATL UNIV, JOHN CURTIN SCH MED RES, DEPT BIOCHEM, CANBERRA, ACT 2601, AUSTRALIA
BHOSALE, SB
;
ROOD, JI
论文数: 0引用数: 0
h-index: 0
机构:
AUSTRALIAN NATL UNIV, JOHN CURTIN SCH MED RES, DEPT BIOCHEM, CANBERRA, ACT 2601, AUSTRALIAAUSTRALIAN NATL UNIV, JOHN CURTIN SCH MED RES, DEPT BIOCHEM, CANBERRA, ACT 2601, AUSTRALIA
ROOD, JI
;
SNEDDON, MK
论文数: 0引用数: 0
h-index: 0
机构:
AUSTRALIAN NATL UNIV, JOHN CURTIN SCH MED RES, DEPT BIOCHEM, CANBERRA, ACT 2601, AUSTRALIAAUSTRALIAN NATL UNIV, JOHN CURTIN SCH MED RES, DEPT BIOCHEM, CANBERRA, ACT 2601, AUSTRALIA
SNEDDON, MK
;
MORRISON, JF
论文数: 0引用数: 0
h-index: 0
机构:
AUSTRALIAN NATL UNIV, JOHN CURTIN SCH MED RES, DEPT BIOCHEM, CANBERRA, ACT 2601, AUSTRALIAAUSTRALIAN NATL UNIV, JOHN CURTIN SCH MED RES, DEPT BIOCHEM, CANBERRA, ACT 2601, AUSTRALIA