Transition state analogs for protocatechuate 3,4-dioxygenase. Spectroscopic and kinetic studies of the binding reactions of ketonized substrate analogs

J. W. Whittaker, J. D. Lipscomb

Research output: Contribution to journalArticle

39 Citations (Scopus)

Abstract

The binding reactions of two heterocyclic analogs of protocatechuate (PCA), 2-hydroxyisonicotinic acid N-oxide and 6-hydroxynicotinic acid N-oxide, to Brevibacterium fuscum protocatechuate 3,4-dioxygenase have been characterized. These analogs were synthesized as models for the ketonized tautomer of PCA which we have previously proposed as the form which reacts with O2 in the enzyme complex (Que, L., Jr., Lipscomb, J.D., Munck, E., and Wood, J.M. (1977) Biochim. Biophys. Acta 485, 60-74). Both analogs have much higher affinity for the enzyme than PCA. Repetitive scan optical spectra of each binding reaction show that at least one intermediate is formed. The spectra of the intermediates are red-shifted (λ(max) = 500 nm) relative to that of native enzyme (λ(max) = 435 nm) but are similar to that of the anaerobic enzyme-PCA complex. In contrast, the spectrum of the final, dead-end complex formed by each analog is significantly blue-shifted (λ(max) < 340 nm) resulting in an apparent bleaching of the chromophore of the enzyme. A transient intermediate exhibiting a similar bleached spectrum has been detected in the enzyme reaction cycle immediately after O2 is added to the enzyme-PCA complex (Bull C., Ballou D.P., and Otsuka, S. (1981) J. Biol. Chem. 256, 12681-12686). Stopped flow measurements of the analog binding reactions show that a relatively weak enzyme complex is initially formed followed by at least two isomerizations leading to the bleached, high affinity complexes. EPR spectra of both the early and final complexes reveal only high spin Fe3+ with negative zero field splitting, showing that the optical bleaching is not due to Fe reduction. The studies show that the ketonized analogs are poor models for the enzyme-substrate complex but do successfully mimic many features of the first oxy complex of the reaction cycle. We propose that substrate ketonization occurs coincident with or after O2 binding and may be involved directly in the O2 insertion reaction.

Original languageEnglish (US)
Pages (from-to)4476-4486
Number of pages11
JournalJournal of Biological Chemistry
Volume259
Issue number7
StatePublished - Jan 1 1984

Fingerprint

Protocatechuate-3,4-Dioxygenase
Kinetics
Substrates
Enzymes
Bleaching
Brevibacterium
Flow measurement
Chromophores
Isomerization
Paramagnetic resonance

Cite this

@article{80c4362eb2e14d5eb51c1de472313e7b,
title = "Transition state analogs for protocatechuate 3,4-dioxygenase. Spectroscopic and kinetic studies of the binding reactions of ketonized substrate analogs",
abstract = "The binding reactions of two heterocyclic analogs of protocatechuate (PCA), 2-hydroxyisonicotinic acid N-oxide and 6-hydroxynicotinic acid N-oxide, to Brevibacterium fuscum protocatechuate 3,4-dioxygenase have been characterized. These analogs were synthesized as models for the ketonized tautomer of PCA which we have previously proposed as the form which reacts with O2 in the enzyme complex (Que, L., Jr., Lipscomb, J.D., Munck, E., and Wood, J.M. (1977) Biochim. Biophys. Acta 485, 60-74). Both analogs have much higher affinity for the enzyme than PCA. Repetitive scan optical spectra of each binding reaction show that at least one intermediate is formed. The spectra of the intermediates are red-shifted (λ(max) = 500 nm) relative to that of native enzyme (λ(max) = 435 nm) but are similar to that of the anaerobic enzyme-PCA complex. In contrast, the spectrum of the final, dead-end complex formed by each analog is significantly blue-shifted (λ(max) < 340 nm) resulting in an apparent bleaching of the chromophore of the enzyme. A transient intermediate exhibiting a similar bleached spectrum has been detected in the enzyme reaction cycle immediately after O2 is added to the enzyme-PCA complex (Bull C., Ballou D.P., and Otsuka, S. (1981) J. Biol. Chem. 256, 12681-12686). Stopped flow measurements of the analog binding reactions show that a relatively weak enzyme complex is initially formed followed by at least two isomerizations leading to the bleached, high affinity complexes. EPR spectra of both the early and final complexes reveal only high spin Fe3+ with negative zero field splitting, showing that the optical bleaching is not due to Fe reduction. The studies show that the ketonized analogs are poor models for the enzyme-substrate complex but do successfully mimic many features of the first oxy complex of the reaction cycle. We propose that substrate ketonization occurs coincident with or after O2 binding and may be involved directly in the O2 insertion reaction.",
author = "Whittaker, {J. W.} and Lipscomb, {J. D.}",
year = "1984",
month = "1",
day = "1",
language = "English (US)",
volume = "259",
pages = "4476--4486",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "7",

}

TY - JOUR

T1 - Transition state analogs for protocatechuate 3,4-dioxygenase. Spectroscopic and kinetic studies of the binding reactions of ketonized substrate analogs

AU - Whittaker, J. W.

AU - Lipscomb, J. D.

PY - 1984/1/1

Y1 - 1984/1/1

N2 - The binding reactions of two heterocyclic analogs of protocatechuate (PCA), 2-hydroxyisonicotinic acid N-oxide and 6-hydroxynicotinic acid N-oxide, to Brevibacterium fuscum protocatechuate 3,4-dioxygenase have been characterized. These analogs were synthesized as models for the ketonized tautomer of PCA which we have previously proposed as the form which reacts with O2 in the enzyme complex (Que, L., Jr., Lipscomb, J.D., Munck, E., and Wood, J.M. (1977) Biochim. Biophys. Acta 485, 60-74). Both analogs have much higher affinity for the enzyme than PCA. Repetitive scan optical spectra of each binding reaction show that at least one intermediate is formed. The spectra of the intermediates are red-shifted (λ(max) = 500 nm) relative to that of native enzyme (λ(max) = 435 nm) but are similar to that of the anaerobic enzyme-PCA complex. In contrast, the spectrum of the final, dead-end complex formed by each analog is significantly blue-shifted (λ(max) < 340 nm) resulting in an apparent bleaching of the chromophore of the enzyme. A transient intermediate exhibiting a similar bleached spectrum has been detected in the enzyme reaction cycle immediately after O2 is added to the enzyme-PCA complex (Bull C., Ballou D.P., and Otsuka, S. (1981) J. Biol. Chem. 256, 12681-12686). Stopped flow measurements of the analog binding reactions show that a relatively weak enzyme complex is initially formed followed by at least two isomerizations leading to the bleached, high affinity complexes. EPR spectra of both the early and final complexes reveal only high spin Fe3+ with negative zero field splitting, showing that the optical bleaching is not due to Fe reduction. The studies show that the ketonized analogs are poor models for the enzyme-substrate complex but do successfully mimic many features of the first oxy complex of the reaction cycle. We propose that substrate ketonization occurs coincident with or after O2 binding and may be involved directly in the O2 insertion reaction.

AB - The binding reactions of two heterocyclic analogs of protocatechuate (PCA), 2-hydroxyisonicotinic acid N-oxide and 6-hydroxynicotinic acid N-oxide, to Brevibacterium fuscum protocatechuate 3,4-dioxygenase have been characterized. These analogs were synthesized as models for the ketonized tautomer of PCA which we have previously proposed as the form which reacts with O2 in the enzyme complex (Que, L., Jr., Lipscomb, J.D., Munck, E., and Wood, J.M. (1977) Biochim. Biophys. Acta 485, 60-74). Both analogs have much higher affinity for the enzyme than PCA. Repetitive scan optical spectra of each binding reaction show that at least one intermediate is formed. The spectra of the intermediates are red-shifted (λ(max) = 500 nm) relative to that of native enzyme (λ(max) = 435 nm) but are similar to that of the anaerobic enzyme-PCA complex. In contrast, the spectrum of the final, dead-end complex formed by each analog is significantly blue-shifted (λ(max) < 340 nm) resulting in an apparent bleaching of the chromophore of the enzyme. A transient intermediate exhibiting a similar bleached spectrum has been detected in the enzyme reaction cycle immediately after O2 is added to the enzyme-PCA complex (Bull C., Ballou D.P., and Otsuka, S. (1981) J. Biol. Chem. 256, 12681-12686). Stopped flow measurements of the analog binding reactions show that a relatively weak enzyme complex is initially formed followed by at least two isomerizations leading to the bleached, high affinity complexes. EPR spectra of both the early and final complexes reveal only high spin Fe3+ with negative zero field splitting, showing that the optical bleaching is not due to Fe reduction. The studies show that the ketonized analogs are poor models for the enzyme-substrate complex but do successfully mimic many features of the first oxy complex of the reaction cycle. We propose that substrate ketonization occurs coincident with or after O2 binding and may be involved directly in the O2 insertion reaction.

UR - http://www.scopus.com/inward/record.url?scp=0021246015&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0021246015&partnerID=8YFLogxK

M3 - Article

C2 - 6323475

AN - SCOPUS:0021246015

VL - 259

SP - 4476

EP - 4486

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 7

ER -