PubMed Health⌕ Search

Biomedical subjects

J W Eckstein

Publications and source records attributed to J W Eckstein.

At least 19 recordsLinked to original sources
PubMed1996

Kinetic analysis of the catalytic domain of human cdc25B.

The Cdc25 cell cycle regulator is a member of the dual-specificity class of protein-tyrosine phosphatases that hydrolyze phosphotyrosine- and phosphothreonine-containing substrates. To study the mechanism of Cdc25B, we have overexpressed and purified the catalytic domain of human Cdc25B (Xu, X., and Burke, S. P. (1996) J. Biol. Chem. 271, 5118-5124). In the present work, we have analyzed the kinetic properties of the Cdc25B catalytic domain using the artificial substrate 3-O-methylfluorescein phosphate (OMFP). Steady-state kinetic analysis indicated that the kcat/Km for OMFP hydrolysis is almost 3 orders of magnitude greater than that for p-nitrophenyl phosphate hydrolysis. Like other dual-specificity phosphatases, Cdc25 exhibits a two-step catalytic mechanism, characterized by formation and breakdown of a phosphoenzyme intermediate. Pre-steady-state kinetic analysis of OMFP hydrolysis indicated that formation of the phosphoenzyme intermediate is approximately 20 times faster than subsequent phosphoenzyme breakdown. The resulting burst pattern of product formation allowed us to derive rate constants for enzyme phosphorylation (26 s-1) and dephosphorylation (1.5 s-1) as well as the dissociation constant for OMFP (0.3 mM). Calculations suggest that OMFP binds with higher affinity and reacts faster with Cdc25B than does p-nitrophenyl phosphate. OMFP is a highly efficient substrate for the dual-specificity protein-tyrosine phosphatases VHR and rVH6, but not for two protein-tyrosine phosphatases, PTP1 and YOP. The ability to observe distinct phases of the reaction mechanism during OMFP hydrolysis will facilitate future analysis of critical catalytic residues in Cdc25 and other dual-specificity phosphatases.

Amino Acid Sequence↗
PubMed1996

Identification of an essential acidic residue in Cdc25 protein phosphatase and a general three-dimensional model for a core region in protein phosphatases.

The reaction mechanism of protein tyrosine phosphatases (PTPases) and dual-specificity protein phosphatases is thought to involve a catalytic aspartic acid residue. This residue was recently identified by site-directed mutagenesis in Yersinia PTPase, VHR protein phosphatase, and bovine low molecular weight protein phosphatase. Herein we identify aspartic acid 383 as a potential candidate for the catalytic acid in human Cdc25A protein phosphatase, using sequence alignment, structural information, and site-directed mutagenesis. The D383N mutant enzyme exhibits a 150-fold reduction in kcat, with Kw only slightly changed. Analysis of sequence homologies between several members of the Cdc25 family and deletion mutagenesis substantiate the concept of a two-domain structure for Cdc25, with a regulatory N-terminal and a catalytic C-terminal domain. Based on the alignment of catalytic residues and secondary structure elements, we present a three-dimensional model for the core region of Cdc25. By comparing this three-dimensional model to the crystal structures of PTP1b, Yersinia PTPase, and bovine low molecular weight PTPase, which share only very limited amino acid sequence similarities, we identify a general architecture of the protein phosphatase core region, encompassing the active site loop motif HCXXXXXR and the catalytic aspartic acid residue.

Amino Acid Sequence↗
PubMed1994

Mechanism-based inhibition of thymidylate synthase by 5-(trifluoromethyl)-2'-deoxyuridine 5'-monophosphate.

Thymidylate synthase (TS) from Lactobacillus casei is inhibited by 5-(trifluoromethyl)-2'-deoxyuridine 5'-monophosphate (CF3dUMP). CF3dUMP binds to the active site of TS in the absence of 5,10-methylenetetrahydrofolate, and attack of the catalytic nucleophile cysteine 198 at C6 of the pyrimidine leads to activation of the trifluoromethyl group and release of fluoride ion. Subsequently, the activated heterocycle reacts with a nucleophile of the enzyme to form a moderately stable covalent complex. Proteolytic digestion of TS treated with [2'-3H]CF3dUMP, followed by sequencing of the labeled peptides, revealed that tyrosine 146 and cysteine 198 are covalently bound to the inhibitor in the enzyme-inhibitor complex. The presence of dithiothreitol (DTT) or beta-mercaptoethanol resulted in the breakdown of the covalent complex, and products from the breakdown of the complex were isolated and characterized. The three-dimensional structure of the enzyme-inhibitor complex was determined by X-ray crystallography, clearly demonstrating covalent attachment of the nucleotide to tyrosine 146. A chemical reaction mechanism for the inhibition of TS by CF3dUMP is presented that is consistent with the kinetic, biochemical, and structural results.

Amino Acid Sequence↗
PubMed1993

Mechanism of bacterial bioluminescence: 4a,5-dihydroflavin analogs as models for luciferase hydroperoxide intermediates and the effect of substituents at the 8-position of flavin on luciferase kinetics.

Bioluminescence catalyzed by bacterial luciferases was measured using FMN, iso-FMN (6-methyl-8-nor-FMN), and FMN analogs carrying the following substituents at position 8: -H, -Cl, -F, SMe, SOMe, -SO2Me, or -OMe. The first-order rate constants for the decay of light emission correlate with the one-electron oxidation potentials of the 4a,5-dihydro forms of the FMN analogs. To determine the values of these potentials, isoalloxazine (flavin) derivatives having the 4a,5-propano-4a,5-dihydro structure and -H, -CH3, -Cl, -OCH3, and -NH2 as substituents at position 8 have been synthesized as models for the 4a-peroxy-4a,5-dihydroflavin intermediates occurring during catalysis by the flavin-dependent monooxygenase luciferase. The tetrahydropyrrole ring between positions 4a and 5 of these isoalloxazine derivatives stabilizes the 4a,5-dihydroflavin by impeding formation of the thermodynamically more stable 1,5-dihydro form. One-electron oxidation potentials (Eobs) were measured by cyclic voltammetry and used to determine the empirical coefficients in the Swain equation. On the basis of this, the one-electron oxidation potentials of 4a,5-propano-4a,5-dihydro analogs with other substituents in position 8 were calculated (Ecalc). The bioluminescence reaction rate is fastest with FMN analogs of lowest oxidation potential; i.e., the slope of the correlation is negative. This indicates that in the rate-limiting step the 4a,5-dihydroflavin moiety donates negative charge. The results are compatible with an intramolecular, chemically initiated electron exchange luminescence mechanism for the bacterial luciferase reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Electrochemistry↗
PubMed1990

A time-dependent bacterial bioluminescence emission spectrum in an in vitro single turnover system: energy transfer alone cannot account for the yellow emission of Vibrio fischeri Y-1.

Yellow fluorescent protein (YFP), which has a bound FMN, was isolated from the marine bacterium Vibrio fischeri strain Y-1b. Its presence in a luciferase [alkanal monooxygenase (FMN-linked); alkanal, reduced-FMN:oxygen oxidoreductase (1-hydroxylating, luminescing), EC 1.14.14.3] reaction mixture causes a striking color change, and an increase in bioluminescence intensity, as well as a faster rate of intensity decay, so that the quantum yield is not changed. The emission spectrum shows two distinct color bands, one at 490 nm attributed to the unaltered emission of the luciferase system, the other peaking in the yellow around 540 nm due to YFP emission. The kinetics of the two color bands differ, so the spectrum changes with time. The yellow emission reaches its initial maximum intensity later than the blue, and then both blue and yellow emissions decay exponentially with nearly the same pseudo-first-order rate constants, linearly dependent on [YFP] (from 0.01 sec-1 with no YFP to a maximum of approximately 0.1 sec-1 at 4 degrees C) but exhibiting a saturation behavior. The data can be interpreted by assuming the interaction of YFP with the peroxyhemiacetal intermediate in the luciferase reaction to form an unstable new complex whose breakdown gives the yellow emitter in its excited state. This simple model fits well the data at [YFP] less than 15 microM. The results indicate that a single primary excited state cannot be responsible for the blue and the yellow emissions.

Bacterial Proteins↗