Search

The L8R and the L8R/152E mutants of 4-OT (4-oxalocrotonate tautomerase) show improved trans-3-chloroacrylic acid dehalogenase (CaaD) activity and diminished 4-OT activity, while the crystal structure of L8R-4-OT is comparable to that of the wild type. These results suggest that the evolution of new functions within the tautomerase superfamily could be quite facile, requiring only a few strategically placed active-site mutations.

A study was conducted on crystal structures of malonate semialdehyde decarboxylate (MSAD), P1A mutant of MSAD, and MSAD inactivated by 3-chloropropiolate to determine mechanism-based inhibitor activated by the hydratase activity of MSAD. The result revealed that a mechanism for the inactivation of MSAD by 3-chloropropiolate could be formulated along with mechanism for the decarboxylase and hydratase activities.

In order to identify active site residues as well as to delineate more fully the differences between trans-3-chloroacrylic acid dehalogenase (CaaD) and cis-CaaD, the (R)- and (S) enantiomers of oxirane-2-carboxylate are investigated as potential irreversible inhibitors. The results help in the crystallographic studies of the inactivated cis-CaaD complex to explain the structural basis for the stereospecific alkylation of this enzyme by (R)-oxirane-2-carboxylate.

Research has been conducted on the hypothetical protein YdcE from Escherichia coli. The crystal structure analysis of this protein demonstrate that it is a dimer and that it has structural diversity within the 4-oxalocrotonate tautomerase family.

Research has been conducted on the amino-terminal proline 4-oxalocrotonate tautomerase. The replacement of Phe-50 with Tyr, Ala, Val and Ile has been performed to investigate the effect of the hydrophobic environment on the pK (sub)a of Pro-1.

The phenylpyruvate tautomerase (PPT) activity of macrophage migration inhibitory factor and its mechanism have been studied with attention to properties of the P1GP1A, Y95F, and N97A mutants. Pro-1 acts as both the general acid and the general base catalyst. PPT has been found to be the same protein as a mammalian cytokine called macrophage migration inhibitory factor (MIF).

Mechanistic and structural study of the 4-oxalocrotonate decarboxylase and vinylpyruvate hydratase complex is presented. Results indicate that two enzymes have been coexpressed.

Macrophage migration inhibitory factor (MIF) exhibits catalytic activity similar to phenylpyruvate tautomerase (PPT) and may be the same protein. The Lys-32 residue in the active site lowers the pK(sub)a of the catalytic base Pro-1.

The biological role of macrophage inhibiting factor (MIF) as an inhibitor of monocyte chemotaxis and random migration was investigated. This was done by examining MIF's ability to act as a tautomerase and by determining the physiological significance of this characteristic. Results showed that MIF inhibits the movement of monocytes when a chemotactic gradient exists. It was also demonstrated that this migration inhibitory property was elicited by the chemotactic gradient of monocyte chemotactic protein 1.

A study reports on the effects of the mutations of active site arginine residues on pH-rate profile and on the pKa values of the general base catalyst Pro-1 in 4-oxalcrotonate tautomerase as determined by direct 15N nuclear magnetic resonance titrations. It is concluded that no arginine residue is responsible for either the essential acid group with pKa of 9.0 in the pH-rate profile or the unusually low pKa of Pro-1. Experimental procedures and results are presented.

The crystal structure of a complex between murine macrophage migration inhibitory factor (MIF) and (E)-2-fluoro-p-hydroxycinnamate, a competitive inhibitor of the tautomerase activity, was determined to 1.8 angstroms resolution. Findings showed that MIF processes the enol isomers more efficiently than the keto isomers mainly because of a decrease in K(sub m). A mechanism is suggested for the MIF-catalyzed tautomerization reaction.

2-Oxo-3-pentynoate was found to be an active-site-directed inhibitor of certain flavoprotein oxidases. Tryptophan 2-monooxygenase is irreversibly inactivated in an active-site-directed manner. The addition of FAD provides no protection from inactivation, while the competitive inhibitor indole-3-acetamide fully protects the enzyme from inactivation. The rate of inactivation indicates saturation kinetics, consistent with the production of a reversible complex between the alkylating agent and the enzyme before inactivation occurs.

The crystal structure of 2-oxo-3-pentynoate-inactivated 4-oxalocrotonate tautomerase was investigated using molecular replacement techniques to determine the mechanism of enzyme inactivation and catalysis. Results reveal that the active site of 4-oxalocrotonate tautomerase is located at a hydrophobic region around the proline residue at position 1. The crystal structure of the enzyme-inhibitor complex was used as the basis for the proposed catalytic reaction.

Expression and purification to homogeneity of 2-oxo-hept-4-ene-1,7-dioate hydratase from Escherichia coli were conducted for kinetic, isotopic, labeling and stereochemical studies. Isotope labeling investigations revealed that the hydratase catalyzes the incorporation of a solvent deuteron at both C-3 and C-5 when the reaction is conducted in 2H2O. The enzymes also catalyzes the exchange of the C-3 proton of the alternate substrate 2-oxo-1,7-heptadioate with solvent deuterons.

The role of the amino-terminal proline in the enzymatic activity catalyzed by macrophage migration inhibitor factor (MIF) was examined through affinity labeling studies with 3-bromopyruvate (3-BP). Results showed that 3-BP acted as the active-site-directed irreversible inhibitor of the enzymatic activity. Moreover, it was also observed that the amino-terminal proline was the only reasonable site for alkylation within the peptide.

The compound 2-oxo-3-pentynoate was synthesized and tested as an inhibitor of the enzyme 4-oxalocrotonate tautomerase from Pseudomonas putida. The enzyme was found to be speedily and irreversibly inactivated by the acetylenic product analogue in a time-dependent manner. It indicated saturation kinetics and was protected from inactivation by the presence of substrate. These findings indicated that inactivation occurs at the active site.

4-oxalocrotonate tautomerase is functional in the isomerization of unconjugated alpha-keto acids. Four mutants of 4-OT have been constructed, where the amino-terminal proline is replaced with glycine, alanine and leucine, resulting in an active enzyme. The quantity of protein obtained only allowed for kinetic analysis. Results indicated that proton transfer between C3 and C5 is partly due to the rigidity of the pyrrolidine ring and the basicity of the secondary amine.

4-Oxalocrotonate tautomerase (4-OT) is an enzyme that catalyzes the isomerization of ketones, with the amino-terminal proline as the catalytic base. A study of the amino-terminal proline's role in isomerization by nuclear magnetic resonance spectroscopy using uniformly 15N-labeled 4-OT revealed that 3-bromopyruvate (3-BP) blocks the amino terminus of 4-OT to Edman degradation. Covalent bonding between a 15N resonance of 4-OT and the methylene carbon of the reduced adduct of 3-BP was also observed. Affinity labeling revealed the stoichiometry of a 4-OT monomer with 3-BP.

The pH dependence of the 4-oxalocrotonate tautomerase (4-OT)-catalyzed isomerization of ketones was examined by measuring the kinetics of 4-OT in different pH levels using 2-hydroxy-2,4-hexadiendioate and 2-hydroxy-2,4-pentadienoate as substrates. The pH dependences of the kinetic parameters for the 4-OT-catalyzed isomerization were bell-shaped with limiting sloped of unity on both sides of the pH optimum. An isomerization mechanism involving general base catalysis by the enzyme is discussed.

The total chemical synthesis of enantiomeric L- and D-4-oxalocrotonate tautomerase (4OT) is described. Structural and stereochemical catalytic activity data indicate that these enzyme fold to form active enzyme complexes and act as efficient catalysts. The enantiomers mostly act on the opposite faces of the dienol substrate to form a noncovalent homohexameric complex. The study uses electrospray ionization time-of-flight mass spectrometry.

The two consecutive reactions in the homoprotocatechuate meta-fission mechanism causing the transformation of 5-(carboxymethyl)-2-hydroxymuconate to 2-oxo-4-heptene-1,7-dioate are catalyzed by the enzymes from Escherichia coli, 5-(carboxymethyl)-2-hydroxymuconate isomerase (CHMI) and 5- (carboxymethyl)-2-oxo-3-hexene-1,6-diolate decarboxylase (COHED). The product of CHMI adopt a R configuration at C-5 which on enzymatic decarboxylation by COHED yield the Z form. In the dienol intermediate the loss of carbon dioxide and involvement of a deuteron occur on the same side.

4-oxalocrotonate decarboxylase (4-OD) and vinylpyruvate hydratase (VPH) from Pseudomonas putida mt-2 are subjected to stereochemical and isotopic labeling studies to determine the conversion of 2-oxo-3-hexenedioate to 2-oxo-4-hydroxypentanoate in the presence of 4-OD/VPH. 4-OD mediates the decarboxylation of the hexenedioate to 2-oxo-4-pentenoate via a dienol intermediate and also converts (5S)-(5-2H) hexenedioate to 4E-(5-2H) hydroxy pentanoate in H2O. Hence, CO2 loss ond deuteron introduction take place on the same side of the dienol intermediate. The OH group C-4 of the hydroxypentanoate is obtained from water, and neither 4-OD nor VPH use a Schiff base intermediate.

Kinetic studies on 4-oxalocrotonate tautomerase (4-OT) reveal that this enzyme is an isomerase which catalyzes the change of (E)-2-oxo-4-hexenedioate to (E)-2-oxo-3-hydroxymuconate, while 2-hydroxymuconate acts as an intermediary substance. 4-OT also ketonizes 4-pentadienoate stereoselectively to (3R)-2-oxo-(3-2H)-4 pentenoate. These results reveal that 4-OT uses a single-base mechanism.

A UV/NMR study of the ketonization of 5-(carboxymethyl)-2-hydroxymuconate (CHM) by its isomerase (CHMI) reveals their behavior vis-a-vis those of 2-hydroxymuconate (HM) and 4-oxalocrotonate tautomerase (4-OT). The study shows that both CHM and HM form a rapid equilibrium between their respective beta,gaamma-enones before their conversion to alpha,beta-enones. However, the partitioning and distribution at equilibrium as well as the stabilities of the beta,gamma-enone intermediates are different. The CHMI intermediate is kinetically capable either way while the 4-OT intermediate is not.

The stereospecificity of 4-oxalocrotonate tautomerase from Pseudomonas putida mt-2 and 5-(carboxymethyl)-2-hydroxymuconate isomerase from Escherichia coli C was determined. The product, 2-oxo-3-(E)-hexenedioate was trapped and processed to (2-2H1)glutaric acid by chemical degradative procedures. It was found that both enzymes ketonize 2-hydroxymuconate stereospecifically to (S)-2-oxo-3-(E)-(5-2H1)hexenedioate.