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Identifying the active site of adeno...

Madhavapeddi, Prashanti.

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Identifying the active site of adenosylcobalamin-dependent glutamate mutase.

Record Type:	Language materials, printed : Monograph/item
Title/Author:	Identifying the active site of adenosylcobalamin-dependent glutamate mutase./
Author:	Madhavapeddi, Prashanti.
Description:	151 p.
Notes:	Chair: E. Neil G. Marsh.
Contained By:	Dissertation Abstracts International63-02B.
Subject:	Biology, Microbiology. -
Online resource:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3042122
ISBN:	0493557083

Identifying the active site of adenosylcobalamin-dependent glutamate mutase.
Madhavapeddi, Prashanti.

Identifying the active site of adenosylcobalamin-dependent glutamate mutase. - 151 p.

Chair: E. Neil G. Marsh.

Thesis (Ph.D.)--University of Michigan, 2002.

Glutamate mutase is an adenosylcobalamin-dependent enzyme that catalyzes the isomerization of (S)-glutamate to (2S, 3S)-3-methylaspartate, a carbon skeleton rearrangement involving radical intermediates. The primary focus of the work presented in this thesis is on identifying the role of two residues: histidine-16 (S subunit) and glutamate-171 (E subunit) of glutamate mutase

ISBN: 0493557083Subjects--Topical Terms:

1017734
Biology, Microbiology.

Identifying the active site of adenosylcobalamin-dependent glutamate mutase.
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020 $a 0493557083
035 $a (UnM)AAI3042122
035 $a AAI3042122
040 $a UnM $c UnM
100 1 $a Madhavapeddi, Prashanti. $3 1259017
245 1 0 $a Identifying the active site of adenosylcobalamin-dependent glutamate mutase.
300 $a 151 p.
500 $a Chair: E. Neil G. Marsh.
500 $a Source: Dissertation Abstracts International, Volume: 63-02, Section: B, page: 0787.
502 $a Thesis (Ph.D.)--University of Michigan, 2002.
520 $a Glutamate mutase is an adenosylcobalamin-dependent enzyme that catalyzes the isomerization of (S)-glutamate to (2S, 3S)-3-methylaspartate, a carbon skeleton rearrangement involving radical intermediates. The primary focus of the work presented in this thesis is on identifying the role of two residues: histidine-16 (S subunit) and glutamate-171 (E subunit) of glutamate mutase
520 $a The MutS subunit contains a conserved histidine residue, His-16, that coordinates the cobalt of adenosylcobalamin in the holo-enzyme. His-16 forms a hydrogen bond with Asp-14 that is important for both coenzyme binding and catalysis. 1-D <super>1</super>H-NMR experiments were performed to measure the first p<italic>K</italic>a of His-16 for wild-type MutS protein and for mutants in which Asp-14 is changed. The p<italic>K</italic>a is characteristic of a solvent-exposed histidine and is very little altered by mutations at Asp-14 or by denaturation of the protein with urea. The results presented indicate that the histidine-aspartate hydrogen bond is not formed in the absence of coenzyme, suggesting that the protein may change conformation upon binding adenosylcobalamin. The NMR structure of MutS and the crystal structure of the holoenzyme of glutamate mutase, obtained subsequently, establish this to be true.
520 $a The solution of the crystal structure of glutamate mutase allowed us to begin to investigate how individual active site residues contribute to catalysis. Several sterically and functionally conservative mutations were introduced at position 171 of the E subunit. In the Glu171Gln mutant, <italic> k</italic>cat is reduced 50-fold, although the <italic>K</italic> m for glutamate is affected only slightly. The activity (<italic>V</italic> max) of the wild-type enzyme is dependent on pH (pKa &sim; 6.6) whereas that of the mutant is independent of pH. The steady-state deuterium kinetic isotope effect is smaller in the mutant enzyme than in the wild-type enzyme, but the steady-state concentration of free radical species on the enzyme is unaffected by the mutation. Rapid reaction kinetics demonstrated that glutamate-171 significantly contributes to substrate binding, as revealed by the higher apparent <italic>K</italic>d values of both substrates for the mutant enzyme when compared to the wild-type enzyme. The properties of the various glutamate-171 mutant enzymes suggest that glutamate-171 serves as an active site base and is very likely involved in stabilizing the radical intermediates.
590 $a School code: 0127.
650 4 $a Biology, Microbiology. $3 1017734
650 4 $a Chemistry, Biochemistry. $3 1017722
690 $a 0410
690 $a 0487
710 2 0 $a University of Michigan. $3 777416
773 0 $t Dissertation Abstracts International $g 63-02B.
790 $a 0127
790 1 0 $a Marsh, E. Neil G., $e advisor
791 $a Ph.D.
792 $a 2002
856 4 0 $u http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3042122