Linked Life Data

9890908

Source:http://linkedlifedata.com/resource/pubmed/id/9890908

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
1
pubmed:dateCreated
1999-2-18
pubmed:abstractText
Orotate phosphoribosyltransferase (OMP synthase, EC 2.4.2.10) forms the UMP precursor orotidine 5'-monophophate (OMP) from orotate and alpha-D-5-phosphoribosyl-1-pyrophosphate (PRPP). Here, equilibrium binding, isotope partitioning, and chemical quench studies were used to determine rate and equilibrium constants for the kinetic mechanism. PRPP bound to two sites per dimer with a KD of 33 microM. Binding of OMP and orotate also occurred to a single class of two sites per dimer, with KD values of 3 and 280 microM, respectively. Pyrophosphate binding to two sites was weak with a KD of 960 microM, and in the presence of bound orotate, its affinity for the first site was enhanced 4-fold (KD = 230 microM). Preformed E.OMP, E.PRPP, E.PPi, and E.orotate complexes were trapped as products in isotope partitioning experiments, indicating that each was catalytically competent and confirming a random mechanism. Rapid quench experiments revealed burst kinetics for product formation in both the forward phosphoribosyltransferase and the reverse pyrophosphorolysis reactions. The steady-state rate in the forward reaction was preceded by a burst (nfwd = 1.5/dimer) of at least 300 s-1. In the pyrophosphorolysis reaction, a burst (nrev = 0.7/dimer; k >/= 300 s-1) was also noted. These results allowed us to develop a complete kinetic mechanism for OPRTase, in which a rapid phosphoribosyl transfer reaction at equilibrium is followed by a slow step involving release of product. When the microviscosity, etarel, of the reaction medium was increased with sucrose, the forward kcat decreased in proportion to etarel with a slope of 0.8. In the reverse reaction a more limited dependence of kcat (slope = 0. 3) was observed. On the basis of the known structures of OPRTase, we propose that a highly conserved, catalytically important, solvent-exposed loop descends during catalysis to shield the active site. In the accompanying paper, the slow product release step is shown to relate to movement of the solvent-exposed loop.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jan
pubmed:issn
0006-2960
pubmed:author
pubmed:issnType
Print
pubmed:day
5
pubmed:volume
38
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
275-83
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
1999
pubmed:articleTitle
Kinetic mechanism of OMP synthase: a slow physical step following group transfer limits catalytic rate.
pubmed:affiliation
Fels Institute for Cancer Research and Molecular Biology, Department of Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S.