Mechanism of reconstitution of brewers' yeast pyruvate decarboxylase with thiamin diphosphate and magnesium

Joseph A. Vaccaro, Edward J. Crane, Thomas K Harris, Michael W. Washabaugh

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

Reconstitution of apo-pyruvate decarboxylase isozymes (PDC, EC 4.1.1.1) from Saccharomyces carlsbergensis was investigated by determination of the steady-state kinetics of the reaction with thiamin diphosphate (TDP) and Mg2+ in the presence and absence of substrate (pyruvate) or allosteric effector (pyruvamide). Reconstitution of the PDC isozyme mixture and α4 isozyme (α4-PDC) exhibits biphasic kinetics with 52 ± 11% of the PDC reacting with k1 = (1.0 ± 0.3) × 10-2 s-1 and 48 ± 12% of the PDC reacting with k2 = (1.1 ± 0.6) × 10-1 s-1 when TDP (KTDP = 0.5 ± 0.2 mM) is added to apo-PDC equilibrated with saturating Mg2+. PDC reconstitution exhibits first-order kinetics with k1 = (1.6 ± 0.5) × 10-2 s-1 upon addition of Mg2+ (KMg2+ = 0.2 ± 0.1 mM) to apo-PDC equilibrated with saturating TDP. Biphasic kinetics for the PDC isozymes provides evidence that apo-PDC reconstitution with TDP and Mg2+ involves two pathways, TDP binding followed by Mg2+ (k1) or Mg2+ binding followed by TDP (k2). This is supported by a change in reconstitution pathway with the order of cofactor addition and is inconsistent with a single pathway involving ordered binding of the metal ion followed by TDP. The presence of pyruvamide has no significant effect on the rate constants for apo-PDC reconstitution and favors the k2 pathway; pyruvate decreases the value of k2 ≤3-fold and has no effect on the value of k1. These results are summarized in a model for apo-PDC reconstitution, and implications for mechanistic studies on PDC are discussed.

Original languageEnglish
Pages (from-to)12636-12644
Number of pages9
JournalBiochemistry
Volume34
Issue number39
StatePublished - Dec 1 1995
Externally publishedYes

Fingerprint

Pyruvate Decarboxylase
Thiamine Pyrophosphate
Yeast
Magnesium
Saccharomyces cerevisiae
Isoenzymes
Kinetics
Pyruvic Acid
Saccharomyces
Metal ions
Rate constants
Metals
Ions
Substrates

ASJC Scopus subject areas

  • Biochemistry

Cite this

Mechanism of reconstitution of brewers' yeast pyruvate decarboxylase with thiamin diphosphate and magnesium. / Vaccaro, Joseph A.; Crane, Edward J.; Harris, Thomas K; Washabaugh, Michael W.

In: Biochemistry, Vol. 34, No. 39, 01.12.1995, p. 12636-12644.

Research output: Contribution to journalArticle

Vaccaro, Joseph A. ; Crane, Edward J. ; Harris, Thomas K ; Washabaugh, Michael W. / Mechanism of reconstitution of brewers' yeast pyruvate decarboxylase with thiamin diphosphate and magnesium. In: Biochemistry. 1995 ; Vol. 34, No. 39. pp. 12636-12644.
@article{6f1626b21b2c44b6884fbf055d8d445d,
title = "Mechanism of reconstitution of brewers' yeast pyruvate decarboxylase with thiamin diphosphate and magnesium",
abstract = "Reconstitution of apo-pyruvate decarboxylase isozymes (PDC, EC 4.1.1.1) from Saccharomyces carlsbergensis was investigated by determination of the steady-state kinetics of the reaction with thiamin diphosphate (TDP) and Mg2+ in the presence and absence of substrate (pyruvate) or allosteric effector (pyruvamide). Reconstitution of the PDC isozyme mixture and α4 isozyme (α4-PDC) exhibits biphasic kinetics with 52 ± 11{\%} of the PDC reacting with k1 = (1.0 ± 0.3) × 10-2 s-1 and 48 ± 12{\%} of the PDC reacting with k2 = (1.1 ± 0.6) × 10-1 s-1 when TDP (KTDP = 0.5 ± 0.2 mM) is added to apo-PDC equilibrated with saturating Mg2+. PDC reconstitution exhibits first-order kinetics with k1 = (1.6 ± 0.5) × 10-2 s-1 upon addition of Mg2+ (KMg2+ = 0.2 ± 0.1 mM) to apo-PDC equilibrated with saturating TDP. Biphasic kinetics for the PDC isozymes provides evidence that apo-PDC reconstitution with TDP and Mg2+ involves two pathways, TDP binding followed by Mg2+ (k1) or Mg2+ binding followed by TDP (k2). This is supported by a change in reconstitution pathway with the order of cofactor addition and is inconsistent with a single pathway involving ordered binding of the metal ion followed by TDP. The presence of pyruvamide has no significant effect on the rate constants for apo-PDC reconstitution and favors the k2 pathway; pyruvate decreases the value of k2 ≤3-fold and has no effect on the value of k1. These results are summarized in a model for apo-PDC reconstitution, and implications for mechanistic studies on PDC are discussed.",
author = "Vaccaro, {Joseph A.} and Crane, {Edward J.} and Harris, {Thomas K} and Washabaugh, {Michael W.}",
year = "1995",
month = "12",
day = "1",
language = "English",
volume = "34",
pages = "12636--12644",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "39",

}

TY - JOUR

T1 - Mechanism of reconstitution of brewers' yeast pyruvate decarboxylase with thiamin diphosphate and magnesium

AU - Vaccaro, Joseph A.

AU - Crane, Edward J.

AU - Harris, Thomas K

AU - Washabaugh, Michael W.

PY - 1995/12/1

Y1 - 1995/12/1

N2 - Reconstitution of apo-pyruvate decarboxylase isozymes (PDC, EC 4.1.1.1) from Saccharomyces carlsbergensis was investigated by determination of the steady-state kinetics of the reaction with thiamin diphosphate (TDP) and Mg2+ in the presence and absence of substrate (pyruvate) or allosteric effector (pyruvamide). Reconstitution of the PDC isozyme mixture and α4 isozyme (α4-PDC) exhibits biphasic kinetics with 52 ± 11% of the PDC reacting with k1 = (1.0 ± 0.3) × 10-2 s-1 and 48 ± 12% of the PDC reacting with k2 = (1.1 ± 0.6) × 10-1 s-1 when TDP (KTDP = 0.5 ± 0.2 mM) is added to apo-PDC equilibrated with saturating Mg2+. PDC reconstitution exhibits first-order kinetics with k1 = (1.6 ± 0.5) × 10-2 s-1 upon addition of Mg2+ (KMg2+ = 0.2 ± 0.1 mM) to apo-PDC equilibrated with saturating TDP. Biphasic kinetics for the PDC isozymes provides evidence that apo-PDC reconstitution with TDP and Mg2+ involves two pathways, TDP binding followed by Mg2+ (k1) or Mg2+ binding followed by TDP (k2). This is supported by a change in reconstitution pathway with the order of cofactor addition and is inconsistent with a single pathway involving ordered binding of the metal ion followed by TDP. The presence of pyruvamide has no significant effect on the rate constants for apo-PDC reconstitution and favors the k2 pathway; pyruvate decreases the value of k2 ≤3-fold and has no effect on the value of k1. These results are summarized in a model for apo-PDC reconstitution, and implications for mechanistic studies on PDC are discussed.

AB - Reconstitution of apo-pyruvate decarboxylase isozymes (PDC, EC 4.1.1.1) from Saccharomyces carlsbergensis was investigated by determination of the steady-state kinetics of the reaction with thiamin diphosphate (TDP) and Mg2+ in the presence and absence of substrate (pyruvate) or allosteric effector (pyruvamide). Reconstitution of the PDC isozyme mixture and α4 isozyme (α4-PDC) exhibits biphasic kinetics with 52 ± 11% of the PDC reacting with k1 = (1.0 ± 0.3) × 10-2 s-1 and 48 ± 12% of the PDC reacting with k2 = (1.1 ± 0.6) × 10-1 s-1 when TDP (KTDP = 0.5 ± 0.2 mM) is added to apo-PDC equilibrated with saturating Mg2+. PDC reconstitution exhibits first-order kinetics with k1 = (1.6 ± 0.5) × 10-2 s-1 upon addition of Mg2+ (KMg2+ = 0.2 ± 0.1 mM) to apo-PDC equilibrated with saturating TDP. Biphasic kinetics for the PDC isozymes provides evidence that apo-PDC reconstitution with TDP and Mg2+ involves two pathways, TDP binding followed by Mg2+ (k1) or Mg2+ binding followed by TDP (k2). This is supported by a change in reconstitution pathway with the order of cofactor addition and is inconsistent with a single pathway involving ordered binding of the metal ion followed by TDP. The presence of pyruvamide has no significant effect on the rate constants for apo-PDC reconstitution and favors the k2 pathway; pyruvate decreases the value of k2 ≤3-fold and has no effect on the value of k1. These results are summarized in a model for apo-PDC reconstitution, and implications for mechanistic studies on PDC are discussed.

UR - http://www.scopus.com/inward/record.url?scp=0028971006&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0028971006&partnerID=8YFLogxK

M3 - Article

VL - 34

SP - 12636

EP - 12644

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 39

ER -