Site-Directed Mutagenesis and Functional Analysis of the Active-Site Residues of the E2 Component of Bovine Branched-Chain α-Keto Acid Dehydrogenase Complex

Menghsiao Meng, David T. Chuang

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Abstract

The catalytic domain of dihydrolipoamide transacylase (E2c) of bovine branched-chain α-keto acid dehydrogenase complex (BCKAD) was overexpressed in Escherichia coli. The E2c catalyzes a reversible acyl transfer reaction between acyl-CoA and dihydrolipoamide, which also occurs spontaneously with a much slower rate. The benzene extracts of both the enzyme-catalyzed and the spontaneous reactions mixture have identical ultraviolet absorbance spectra with a maximum at 233–234 nm, which is characteristic of S-acyldihydrolipoamide. The spontaneous reaction rate of various acyl-CoA is in the order of acetoacetyl-CoA > acetyl-CoA > isobutyryl-CoA > isovaleryl-CoA. In other words, the spontaneous acyl transfer is faster when the substituent (R) of acyl-CoA (R-CO-S-CoA) is a more electron-withdrawing group. This result indicates that a negative charge occurs in the substrate during the acyl transfer process. The function of the active-site histidine (His391) and serine (Ser338) of bovine E2c was analyzed by site-directed mutagenesis. Substitution of His391 or Ser338 with alanine caused drastic decreases in catalytic efficiencies by 3–4 orders of magnitude. The residual activity of H391A increased as the pH of the reaction buffer was elevated. These data support the base-catalyzed mechanism inferred from that of chloramphenicol acetyltransferase (CAT). In this reaction, the active-site histidine acts as a general base, and the active-site serine provides a hydrogen bond to the putative negatively charged tetrahedral transition state. Moreover, when Ala348 was changed to valine, the catalytic efficiency for isovaleryl-CoA decreased about 10-fold, and that for acetyl-CoA increased about 3-fold. Ala348 presumably contacts the isobutyl group of isovaleryl-CoA in the acyl transfer reaction. Our results indicate that this residue plays a key role in the substrate preference of bovine E2c.

Original languageEnglish (US)
Pages (from-to)12879-12885
Number of pages7
JournalBiochemistry
Volume33
Issue number43
DOIs
StatePublished - Nov 1 1994

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3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)
Acyl Coenzyme A
Mutagenesis
Functional analysis
Site-Directed Mutagenesis
Catalytic Domain
Acetyl Coenzyme A
Histidine
Serine
Chloramphenicol O-Acetyltransferase
Valine
Substrates
Coenzyme A
Carbon Monoxide
Benzene
Alanine
Escherichia coli
Reaction rates
Hydrogen bonds
Buffers

ASJC Scopus subject areas

  • Biochemistry

Cite this

@article{4e6e776230eb4b499490c137d859797b,
title = "Site-Directed Mutagenesis and Functional Analysis of the Active-Site Residues of the E2 Component of Bovine Branched-Chain α-Keto Acid Dehydrogenase Complex",
abstract = "The catalytic domain of dihydrolipoamide transacylase (E2c) of bovine branched-chain α-keto acid dehydrogenase complex (BCKAD) was overexpressed in Escherichia coli. The E2c catalyzes a reversible acyl transfer reaction between acyl-CoA and dihydrolipoamide, which also occurs spontaneously with a much slower rate. The benzene extracts of both the enzyme-catalyzed and the spontaneous reactions mixture have identical ultraviolet absorbance spectra with a maximum at 233–234 nm, which is characteristic of S-acyldihydrolipoamide. The spontaneous reaction rate of various acyl-CoA is in the order of acetoacetyl-CoA > acetyl-CoA > isobutyryl-CoA > isovaleryl-CoA. In other words, the spontaneous acyl transfer is faster when the substituent (R) of acyl-CoA (R-CO-S-CoA) is a more electron-withdrawing group. This result indicates that a negative charge occurs in the substrate during the acyl transfer process. The function of the active-site histidine (His391) and serine (Ser338) of bovine E2c was analyzed by site-directed mutagenesis. Substitution of His391 or Ser338 with alanine caused drastic decreases in catalytic efficiencies by 3–4 orders of magnitude. The residual activity of H391A increased as the pH of the reaction buffer was elevated. These data support the base-catalyzed mechanism inferred from that of chloramphenicol acetyltransferase (CAT). In this reaction, the active-site histidine acts as a general base, and the active-site serine provides a hydrogen bond to the putative negatively charged tetrahedral transition state. Moreover, when Ala348 was changed to valine, the catalytic efficiency for isovaleryl-CoA decreased about 10-fold, and that for acetyl-CoA increased about 3-fold. Ala348 presumably contacts the isobutyl group of isovaleryl-CoA in the acyl transfer reaction. Our results indicate that this residue plays a key role in the substrate preference of bovine E2c.",
author = "Menghsiao Meng and Chuang, {David T.}",
year = "1994",
month = "11",
day = "1",
doi = "10.1021/bi00209a020",
language = "English (US)",
volume = "33",
pages = "12879--12885",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "43",

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TY - JOUR

T1 - Site-Directed Mutagenesis and Functional Analysis of the Active-Site Residues of the E2 Component of Bovine Branched-Chain α-Keto Acid Dehydrogenase Complex

AU - Meng, Menghsiao

AU - Chuang, David T.

PY - 1994/11/1

Y1 - 1994/11/1

N2 - The catalytic domain of dihydrolipoamide transacylase (E2c) of bovine branched-chain α-keto acid dehydrogenase complex (BCKAD) was overexpressed in Escherichia coli. The E2c catalyzes a reversible acyl transfer reaction between acyl-CoA and dihydrolipoamide, which also occurs spontaneously with a much slower rate. The benzene extracts of both the enzyme-catalyzed and the spontaneous reactions mixture have identical ultraviolet absorbance spectra with a maximum at 233–234 nm, which is characteristic of S-acyldihydrolipoamide. The spontaneous reaction rate of various acyl-CoA is in the order of acetoacetyl-CoA > acetyl-CoA > isobutyryl-CoA > isovaleryl-CoA. In other words, the spontaneous acyl transfer is faster when the substituent (R) of acyl-CoA (R-CO-S-CoA) is a more electron-withdrawing group. This result indicates that a negative charge occurs in the substrate during the acyl transfer process. The function of the active-site histidine (His391) and serine (Ser338) of bovine E2c was analyzed by site-directed mutagenesis. Substitution of His391 or Ser338 with alanine caused drastic decreases in catalytic efficiencies by 3–4 orders of magnitude. The residual activity of H391A increased as the pH of the reaction buffer was elevated. These data support the base-catalyzed mechanism inferred from that of chloramphenicol acetyltransferase (CAT). In this reaction, the active-site histidine acts as a general base, and the active-site serine provides a hydrogen bond to the putative negatively charged tetrahedral transition state. Moreover, when Ala348 was changed to valine, the catalytic efficiency for isovaleryl-CoA decreased about 10-fold, and that for acetyl-CoA increased about 3-fold. Ala348 presumably contacts the isobutyl group of isovaleryl-CoA in the acyl transfer reaction. Our results indicate that this residue plays a key role in the substrate preference of bovine E2c.

AB - The catalytic domain of dihydrolipoamide transacylase (E2c) of bovine branched-chain α-keto acid dehydrogenase complex (BCKAD) was overexpressed in Escherichia coli. The E2c catalyzes a reversible acyl transfer reaction between acyl-CoA and dihydrolipoamide, which also occurs spontaneously with a much slower rate. The benzene extracts of both the enzyme-catalyzed and the spontaneous reactions mixture have identical ultraviolet absorbance spectra with a maximum at 233–234 nm, which is characteristic of S-acyldihydrolipoamide. The spontaneous reaction rate of various acyl-CoA is in the order of acetoacetyl-CoA > acetyl-CoA > isobutyryl-CoA > isovaleryl-CoA. In other words, the spontaneous acyl transfer is faster when the substituent (R) of acyl-CoA (R-CO-S-CoA) is a more electron-withdrawing group. This result indicates that a negative charge occurs in the substrate during the acyl transfer process. The function of the active-site histidine (His391) and serine (Ser338) of bovine E2c was analyzed by site-directed mutagenesis. Substitution of His391 or Ser338 with alanine caused drastic decreases in catalytic efficiencies by 3–4 orders of magnitude. The residual activity of H391A increased as the pH of the reaction buffer was elevated. These data support the base-catalyzed mechanism inferred from that of chloramphenicol acetyltransferase (CAT). In this reaction, the active-site histidine acts as a general base, and the active-site serine provides a hydrogen bond to the putative negatively charged tetrahedral transition state. Moreover, when Ala348 was changed to valine, the catalytic efficiency for isovaleryl-CoA decreased about 10-fold, and that for acetyl-CoA increased about 3-fold. Ala348 presumably contacts the isobutyl group of isovaleryl-CoA in the acyl transfer reaction. Our results indicate that this residue plays a key role in the substrate preference of bovine E2c.

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