Linked Life Data

15882057

Source:http://linkedlifedata.com/resource/pubmed/id/15882057

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
19
pubmed:dateCreated
2005-5-10
pubmed:abstractText
(4-Hydroxyphenyl)pyruvate dioxygenase (HPPD) catalyzes the conversion of (4-hydroxyphenyl)pyruvate (HPP) to homogentisate (HG). This reaction involves decarboxylation, substituent migration, and aromatic oxygenation in a single catalytic cycle. HPPD is a unique member of the alpha-keto acid dependent oxygenases that require Fe(II) and an alpha-keto acid substrate to oxygenate or oxidize an organic molecule. We have examined the reaction coordinate of HPPD from Streptomyces avermitilis using rapid mixing pre-steady-state methods in conjunction with steady-state kinetic analyses. Acid quench reactions and product analysis of homogentisate indicate that HPPD as isolated is fully active and that experiments limited in dioxygen concentration with respect to that of the enzyme do involve a single turnover. These experiments indicate that during the course of one turnover the concentration of homogentisate is stoichiometric with enzyme concentration by approximately 200 ms, well before the completion of the catalytic cycle. Subsequent single turnover reactions were monitored spectrophotometrically under pseudo-first-order and matched concentration reactant conditions. Three spectrophotometrically distinct intermediates are observed to accumulate. The first of these is a relatively strongly absorbing species with maxima at 380 and 480 nm that forms with a rate constant (k(1)) of 7.4 x 10(4) M(-)(1) s(-)(1) and then decays to a second intermediate with a rate constant (k(2)) of 74 s(-)(1). The rate constant for the decay of the second intermediate (k(3)) is 13 s(-)(1) and is concomitant with the formation of the product, homogentisate, based on rapid quench and pre-steady-state fluorescence measurements. The rate constant for this process decreases to 7.6 s(-)(1) when deuterons are substituted for protons in the aromatic ring of the substrate. The release of product from the enzyme is rate limiting and occurs at 1.6 s(-)(1). This final event exhibits a kinetic isotope effect of 2 with deuterium oxide as the solvent, consistent with a solvent isotope effect on V(max) of 2.6 observed in steady-state experiments.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
May
pubmed:issn
0006-2960
pubmed:author
pubmed:issnType
Print
pubmed:day
17
pubmed:volume
44
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
7189-99
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
2005
pubmed:articleTitle
Accumulation of multiple intermediates in the catalytic cycle of (4-hydroxyphenyl)pyruvate dioxygenase from Streptomyces avermitilis.
pubmed:affiliation
Department of Chemistry and Biochemistry, University of Wisconsin-Milwaukee, 3210 North Cramer Street, Milwaukee, Wisconsin 53211-3029, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S., Research Support, N.I.H., Extramural