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Phosphatidylinositol-specific phospholiphase C accepts a variety of alkyl and aryl inositol phosphates as substrates, making it a suitable enzyme for studying mechanism of phosphoryl transfer by probing the linear free-energy relationship. A study of Bronsted-type linear free-energy relationship (LFER) relationship to compare mechanisms of enzymatic and nonenzymatic reactions is conducted to assess the role of hydrophobicity in the leaving group as a general acid-enabling factor.

Research demonstrates that placing 2-aminopurine one base below the nascent base pair greatly enhances the signal intensity while placing it two bases below provides acceptable signal-to-noise ratio. The research findings are useful in the determination of kinetic constants for dNTP and catalytic magnesium incorporation, which is facilitated by DNA polymerase beta.

The established concept of high nucleotide incorporation fidelity by DNA polymerases suggests a new 'simplified' model. Research points out that the fidelity arises from differential substrate binding step at the transition state, which facilitates substrate selectivity by DNA polymerases.

Research has been conducted on the oncogenic protein gankyrin, containing ankyrin repeats. Results indicate that this protein is involved in the degradation and phosphorylation of the retinoblastoma gene product Rb.

The authors have evaluated the conceptual and analytical progress made since the publication of the pancreatic secreted phospholipase A (sub)2 survey. The purpose of this review is to focus on the methods of determining the parameters describing the interfacial turnover cycle events useful for the structure-function correlation for the specific reaction steps.

Research has been conducted on the stereoselectivity of the DNA polymerase. The pre-steady-state kinetic analysis of this stereoselectivity and its alteration by metal ions and by site-directed mutagenesis are discussed.

The Arg69 residue in phosphatidylinositol-specific phospholipase C not only binds the phosphate but positions it close to the 2-OH group of inositol in proper alignment for nucleophilic attack. The bidentate nature of this residue produces the large thio effects and stereoselectivity seen in this enzyme.

The Asp33, Arg69, and His32 residues of phosphatidylinositol-specific phospholipase C forms a triad that activates the phosphate group undergoing nucleophilic attack and protonates the leaving group. The formation of the triad depends on hydrophobic interactions between the enzyme and the leaving group.

Research on an intermediate structure of DNA polymerase beta shows that there is no rate-limiting step during conformational change. This enzyme has two metal-binding sites, one at the dNTP binding site and one at the catalytic site.

The catalytic functions of amino acid residue in the structure-function relationship of DNA polymerase beta are examined. Results indicate that while Lys280, Asn294, and Glu295 mediate positioning or stabilizing the template DNA, Arg149, Ser180, Arg183, and Ser188 are presumably involved in the nonspecific interactions between polymerase and the triphosphate moiety.

Research has been conducted on the pancreatic phospholipase A (sub)2. Results indicate that this phospholipase demonstrates strong preference for the binding to the anionic interface.

A structure-function analysis of a tumor suppressor p16 (super)INK4A homologue is presented. Characterization of mutant conformations by NMR is discussed.

Studies were conducted to determine the structural basis for the anion-induced k*cat activation. Results show that this activation lies predominantly in cationic residues K53 and K56 of the pancreatic phospholipase A2 (PLA2). Structures of these residues were virtually identical to the crystal structure of K56M even if 22% of the residues are different in bovine and porcine PLA2. Data also suggest that K53 and K56 must play a pivotal role in k*cat activation of WT by the anionic charge at the interface, without a substantial effect on the other parameters.

The mechanism of phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis was investigated. Site-directed mutagenesis was used to examine the functional and structural roles of His-32, His-82, Asp -274 and Asp-33. Results show that His-32 acts as a general base to abstract the proton from 2-OH and induces the attack of the deprotonated 2-oxygen on the atom of the phosphorus. His-82, on the other hand, functions as the general acid with support from Asp-33.

Conformational changes in the catalytic cycle of DNA polymerase beta were determined by stopped-flow fluorescence assay, utilizing a synthetic DNA primer/template with 2-aminopurine (2-AP) at the template position opposite the incoming dNTP. Two phases of fluorescence change were found in the stopped-flow fluorescence assay of the incorporation of the correct nucleotide dTTP. The rates of the two phases and their dependence on (dTTP) and (Mg2+) indicate that the fast conformational change is caused by the binding of MgdNTP and the slow conformational change is caused by the binding of the catalytic Mg2+ ion.

The detailed kinetic characterization of phosphatidylinositol-specific phospholipase C (PI-PLC) as well as the role of Arg-69 in the catalysis were determined. (2R)-1,2-dipalimitoyloxypropanethiophospho-1-D-myoinositol (DPsPI) was utilized for assaying PI-PLC. Results have shown that Arg-69 was needed for the enzyme catalysis by stabilizing the negative charge on the pentacoordinate phosphate resulting to a lower level of activity compared to that of wild types.

The triple mutant bovine pancreatic phospholipases A2 (PLA2), Y52,73F/D99N, was analyzed to determine the role of the conserved structural water in hydrogen bonding to the catalytic triad in aspartate99 (D99) mutants. The detailed analysis of the conformational structure of Y52,73F/D99N indicated the role of D99 in the biochemical function of PLA2. The triple mutant PLA2 also exhibited an altered hydrogen-bonding network which linked the catalytic active site to the interfacial binding region.

The kinetic parameters for all possible correct and incorrect pairing between the A, T, G and C bases were obtained for wild-type (WT) rat DNA polymerase beta (pol beta) and the R283A mutant under pre-steady-state kinetic assay conditions. Results indicated that for both WT and R283A, the fidelity is determined primarily by kpol (decreases for the incorporation of incorrect nucleotides). In general, the fidelity for the Y*X mismatch is different from that of the X*Y mismatch. It was also determined that the fidelity of R283A is reduced in all 12 mispairs compared to that of WT and the decreases in the fidelity of the R283A mutant are mainly due to alterations in the values of kpol.

The structural, binding, interfacial and catalytic properties of bovine pancreatic phospholipase A2 (PLA2) expressed in Escherichia coli were characterized upon multiple mutation and deletion of its C-terminal segment, respectively. The results showed that the uncoupling between the calcium and substrate substantially lowers the affinity of the deletion mutant for active site-directed ligands on zwitterionic interfaces.

Reverse transesterification reaction by the phosphatidylinositol-specific phospholipase (PI-PLC) catalysis was utilized to produce O-alkyl inositol 1-phosphates. Cleavage of phosphatidylinositol from soybean phospholipids was induced by the single-step catalysis of PI-PLC. The reaction produced a mixture of phosphorylated derivatives such as 1,2-cyclic phosphate, inositol 1-phosphate, 4- and 5-phosphorylated cogeners. PI-PLC synthesis can also be applied to complex compounds of alkyl inositol phosphodiesters to attach inositol phosphate residue to carbohydrates nucleosides and peptides.

Tumor suppressor p16(sup INK4A), with eight N-terminal amino acids deleted (p16/delta 1-8), is cloned, expressed in Escherichia coli and purified for the analysis of the structure and function of p16. The protein has low solubility, high aggregation tendency, and low stability. Its secondary structure is highly alpha-helical and global conformation is very flexible. p16 has four ankyrin repeats that are present in helix-turn-helix structure. Site directed mutants, P114L and G101W, cause global conformational changes, and D84H shows increased aggregation tendency.

The kinetics of DNA polymerase (pol) beta were investigated by rapid-chemical-quench and DNA trapping experiments. The results revealed that catalysis by pol beta is progressive and begins rapidly under pres-steady state conditions. However, pol beta had low fidelity and processivity as compared to other polymerases. Meanwhile, site-directed mutagenesis revealed that arginine-283 is not important to the stability and function of DNA pol beta.

The contributions of each of the seven disulfide bonds of bovine pancreatic phospholipase A2 to the structure, conformational stability and catalytic function of the enzyme have been determined. The results showed that only the disulfide C84-C96 is critically important to the folding pathway of PLA2 while the disulfide C11-C77 is most important to the conformational stability of the enzyme. The mutant C29A-C45A was found to exhibit the greatest decrease in K(sub cat,app) and the greatest increase in K(sub m,app) toward DC8PC micelles.

Examination of the structure and functions of a nonconservative mutant and a conservative mutant of muscle adenylate kinase (AK) by spectroscopic studies reveals that Lys21 facilitates catalysis by orienting the triphosphate chain of MgATP to a proper conformation. The transition state in the catalysis of AK is stabilized by the side chain of Lys 21. The interactions between the active site residues and the substrates are controlled by the interaction of Lys21 with the phosphate chain.

High resolution NMR studies of the enzymatic catalysis by phospholipase A2 (PLA2), and mutagenesis of the active aspartate-49 (Asp-49) site residue indicate the necessity for the presence of Asp-49 for catalysis by PLA2. Five mutant enzymes resulting from Asp-49 enable binding and orientation of the Ca2+ needed for catalytic activity. Substituents affect conformation of the mutants locally but do not influence the overall conformation.

The isolation of 1L-configuration of naturally occurring chiro-inositol phospholipids has led to the identity of both the enantiomers of chiro-inositol in human biological fluids which have stereochemical similarity. PI-PLC cleaves both 1D-myo and 1L-chiro configurations of phosphatidylinositols suggesting that 1L-chiro-inositol is metabolized by enzymes amenable to myo-inositol.

A study was conducted on the structural and functional effects of phenylalanine-22 (Phe-22) and phenylalanine-106 (Phe-106) aromatic sandwich in phospholiphase A2. Proton nuclear Magnetic resonance and denaturation were used to characterize the mutants. Results showed that neitherthe Phe-106 face-to-face pair nor the Phe-22/Tyr-111 edge-to-face pair significantly affects PLA2 structure. Results also showed that aromaticity was not a significant factor in the function of the residues. However, the hydrophobic bulk of the residues were found to be important for catalysis.

The nick ligation capabilities of the African swine fever virus (ASFV)-encoded DNA ligase and reported the first complete 3' fidelity analysis, employing catalytic parameters, foe any DNA ligase is examined. The results indicate that ASFV DNA ligase is the lowest-fidelity DNA ligase reported, capable of ligating a 3' C:T mismatched nick more efficiently than nicks containing Watson-Crick base pairs.

The hypothesis that nonconserved residues help in finding out the ligand specificity of yeast Rad53 FHA domains at the +3 position is tested. Results based on library screening, binding analyses and structural analyses using NMR indicate the importance of the residues for the structural integrity of the loops.

Results reveal that in the binding of the nuclear factor kappaB inhibitor, IkappaBalpha with the nuclear factor, the contacts facilitating binding are located within the last two terminal ankyrin repeats. Further, the loop region involved in the binding and the first four N-terminal ankyrin repeats are responsible for cyclin-dependent kinase 4 binding and inhibition.

Research has been conducted on human T-cell leukemia virus 1 infected cells. The authors describe the route regulating CDK4 activity by Tax oncoprotein in human T-cell leukemia virus 1 infected cells.

Research demonstrates that pancreatic IB phospholipase A(sub)2 forms large aggregates with monodisperse alkyl sulfates with amphiphile molecules binding along their polar headgroup to the interface (i-face) binding region of phospholipase A(sub)2. The complex, E(sup)#, self-aggregates and sticks to hydrophobic surfaces. Furthermore, E(sup)# is formed without catalytic cofactor calcium or an active-site ligand.

Research describes engineering of a metal-independent bacterial phosphatidylinositol-specific phospholipase C into a metal-dependent enzyme through mutagenesis at the R69D site. The kinetic analysis shows activation of the catalytic activity by calcium significantly. Furthermore, calcium is required to restore the catalytic site microenvironment as shown by NMR studies.

A novel Ca(super 2plusmn) dependent Streptomyces antibioticus PI-PLC (saPLC1) and its mutants display unprecedented magnitudes of thio effects, reverse thio effects, and Rp/Sp stereoselectivity is reported. The findings demonstrate that the magnitudes of thio effects in enzymatic reactions cannot be assumed to fall within the narrow range defined by nonenzymatic reactions.

A DNA polymerase is reported, with specificity for five base pairs.

The identification of a novel Arg-Asp-His catalytic triad that has functions in phosphate activation and leaving group protonation to catalyze the P-O bond cleavage for phosphatidylinositol-specific phospholipase C (PI-PLC) from B. thuringiensis is reported. Results showed that Asp33 and Arg69 function inseparably. In addition, communication between Arg69 and His82 was found to be disrupted in the PI-PLC mutant D33A.

The functional roles of two divalent metal ions in the catalysis by Pol beta from rat brain were examined, using Cr(III)dTTP and stopped-flow fluorescence. While it has been established that DNA polymerases require metal ions for catalysis, there has been no kinetic evidence that DNA polymerases require two metal ions and the functional roles of these two metal ions have not been investigated. It was found that two metal ions are important for the fidelity of Pol beta, with results showing that one metal ion is required for each of the two conformational change steps.

Substitution of a phosphoryl oxygen with sulfur is a technique widely used in enzyme and ribozyme studies. The ratio kolks for substitution of nonbridging oxygen and a bridging oxygen as type I and II thio effects, have been defined. The thio effects and site-directed mutagenesis are used to support the mechanism for phosphatidylinositol-specific phospholipase C (PI-PLC).

The first affirmation of reversal of a thio effect for phosphatidylinositol-specific phospholipase C upon single amino acid substitution is reported. The thio effect refers to the ratio k(sub O)/k(sub S) where O represents a normal oxygen substrate while S stands for a sulfur analog. A reversal of type II thio effect, or the thio effect of phosphorothiolates, occurs by a single mutation, while type I thio effects, or the thio effect of phosphorothioates, have been observed in the range of 1-1000, but most occur within 10-100.

Primary rate and equilibrium factors for 60 site-directed mutants of bovine pancreatic phospholipase A2 (PLA2) were examined to allow the analysis of incremental contributions of the substitution of specific residues. Findings indicated that the interfacial recognition region of PLA2 imposes an allosteric regulation of the substrate binding and chemical step because of the plasticity of certain segments and domains.