What question did this study set out to answer?

To examine the structural and mechanistic role of Asp 270 in regulating the pKa and catalytic function of His 297 in mandelate racemase from Pseudomonas putida.

January 1, 1996Biochemistry

Mechanism of the Reaction Catalyzed by Mandelate Racemase: Structure and Mechanistic Properties of the D270N Mutant,

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Authors

AKAbraham T. KallarakalGeorgetown University BMBharati MitraWayne State University JKJohn W. KozarichBoston College

Discussion

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Implication

Biochemical analysis demonstrates disrupted histidine catalysis in mandelate racemase D270N mutants, indicating that Asp 270 and His 297 function as a catalytic dyad.

Key Points

To examine the structural and mechanistic role of Asp 270 in regulating the pKa and catalytic function of His 297 in mandelate racemase from Pseudomonas putida.
Engineered the D270N mutant of mandelate racemase and solved its high-resolution crystal structure complexed with the inhibitor (S)-atrolactate.
Evaluated catalytic activity and pH-dependent kcat profiles for the racemization of (R)- and (S)-mandelates.
Assayed alpha-proton exchange with solvent and stereospecific elimination using substrate analogues to probe residue-specific catalysis.
The D270N substitution reduced kcat values by approximately 10,000-fold for both (R)- and (S)-mandelate substrates.
Crystallographic analysis showed active site geometry remained largely preserved, with the side chain of His 297 tilted and shifted by approximately 0.5 Å away from Asn 270.
The pH-dependent kinetic profile in the (R)-to-(S) direction lost its ascending limb, exhibiting an apparent pKa shift of the His 297 conjugate acid to approximately 10 while retaining stereospecific exchange only for (S)-mandelate.

Cite This Study

Kallarakal et al. (1996) studied this question.

synapsesocial.com/papers/6a9e76cb065205258095ea1a https://doi.org/10.1021/bi960174m

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January 1, 1996Biochemistry

Mechanism of the Reaction Catalyzed by Mandelate Racemase: Structure and Mechanistic Properties of the D270N Mutant,

View Full Paper

Ask AI

Bookmark

Authors

AKAbraham T. KallarakalGeorgetown University BMBharati MitraWayne State University JKJohn W. KozarichBoston College

Discussion

Member takes

Implication

Biochemical analysis demonstrates disrupted histidine catalysis in mandelate racemase D270N mutants, indicating that Asp 270 and His 297 function as a catalytic dyad.

Key Points

To examine the structural and mechanistic role of Asp 270 in regulating the pKa and catalytic function of His 297 in mandelate racemase from Pseudomonas putida.
Engineered the D270N mutant of mandelate racemase and solved its high-resolution crystal structure complexed with the inhibitor (S)-atrolactate.
Evaluated catalytic activity and pH-dependent kcat profiles for the racemization of (R)- and (S)-mandelates.
Assayed alpha-proton exchange with solvent and stereospecific elimination using substrate analogues to probe residue-specific catalysis.
The D270N substitution reduced kcat values by approximately 10,000-fold for both (R)- and (S)-mandelate substrates.
Crystallographic analysis showed active site geometry remained largely preserved, with the side chain of His 297 tilted and shifted by approximately 0.5 Å away from Asn 270.
The pH-dependent kinetic profile in the (R)-to-(S) direction lost its ascending limb, exhibiting an apparent pKa shift of the His 297 conjugate acid to approximately 10 while retaining stereospecific exchange only for (S)-mandelate.

Cite This Study

Kallarakal et al. (1996) studied this question.

synapsesocial.com/papers/6a9e76cb065205258095ea1a https://doi.org/10.1021/bi960174m

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