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The development of a new carbonylation reaction of alkynes with 2-bromophenylboronic acid leading to the formation of indenones is reported. The results suggest that while active site interactions at the leaving group oxygen atom are similar for phosphate and sulfate esters, the interactions with the nonbridging oxygen atom differ significantly.

The DNA repair exhibits an evolvable system, allowing for flexibility in recruiting any enzyme to a new function, so that gene duplication in a redundant DNA repair activity would not be required. Most DNA repair enzymes have broad substrate specificity, enabling an abundant source of enzymes with low promiscuity to simplify DNA repair activity.

Alkaline phosphatase-catalyzed hydrolysis of alkyl phosphates reveal the ionizable groups involved in the binding and catalysis. The enzyme kinetics indicate that the inorganic phosphate dianion seem to bind to the highly charged alkaline phosphatase active site as determined by a new (sup)32P-based assay.

Research shows that Escherichia coli alkaline phosphatase possesses a low level of phosphodiesterase activity. Active site's arginine mutation to serine reduces monoesterase activity without affecting diesterase activity. Results point out to a divergent evolution of phosphodiesterases and phosphomonoesterases in the alkaline phosphatase superfamily.

The application of the human alkyladenine DNA glycosylase for the N-glycosyl bond formation is discussed. The observed DNA glycolase is shown to be extremely beneficial, as it allows the glycolase to bypass the base excision repair pathway.

A variety of symmetric and asymmetric lesion-containing oligonucleotides are used to characterize the apurinic/apyrimidinic site endonuclease 1 (APE1) stimulation of alkyladenine DNA glycosylase alkyladenine DNA glycosylase (AAG). The results proposed that nonspecific DNA binding interactions facilitate transient exposure of the abasic site so that it could be captured by APE1.

The approach employs a DNA repair glycosylase for the identification of the unpaired nucleotides that are normally used for the recognition and repair of DNA damage. The duplex stability and catalytic efficiency of the nucleotides are shown to exhibit an inverse correlation between them.

The natural fluorescence of the 1,[N.sup.6]-ethenoadenosine ([epsi]A) lesion is utilized to perform kinetic analysis of binding, nucleotide flipping, base excision, and product release by human alkyladenine DNA glycosylase which initiated the repair of a wide variety of alkylated and deaminated purine lesions in DNA. The highly favorable equilibrium for flipping of [epsi]A ensured the sequence context independent elimination of [epsi]A which is highly efficient despite the relatively slow rate of N-glycosidic bond hydrolysis.

An oligonucleotide substrate containing two sites of DNA damage was used to gain insight into the mechanism by which human alkyladenine DNA glycosylase (AAG) has located sites of damage. The findings revealed that AAG has the ability to slide for short distances along DNA at physiological salt concentrations and the diffusion on undamaged DNA has contributed to the search for sites of DNA damage.

The studies of the active site arginine of Escherichia coli alkaline phosphatase which contributes to catalysis of the phosphoryl transfer reaction both through ground state binding interactions and through specific stabilization on the transition state is reported. The results suggested that the guanidinium group engages in hydrogen-bonding interactions that achieve optimal geometry in the transition state of the reaction.

A series of alkaline phosphates (AP) substrates with similar geometries but systematically varying charge on the nonbridging oxygen atom is investigated to determine the energetic properties of the electrostatic interaction between the Zn(super 2+) bimetallocluster and the nonbridging oxygen atom. There is electrostatic repulsion between the two Zn(super 2+) ions bound only 4 angstrom apart and this close spacing within the active site renders it difficult to effectively solvate the metallocluster.

Human alkyladenine DNA glycosylase (AAG) use in acid-base catalysis for selective excision of damaged purines is examined. The prominent role of nucleobase protonation in catalysis by AAG provides a rationale for its specialization toward damaged purines while effectively excluding pyrimidines.

The bonding properties of the phosphoryl group are examined via vibrational spectroscopy for a series of compounds in which the phosphoryl substituent was varied systematically and extensively. It was suggested that ground-state distortions of substrates bound to enzymes could provide readout of the electrostatic active site environment.

The active site arginine of alkaline phosphatase did not make major changes to the leaving group dependence.

A functional relationship was observed in an investigation of whether alkaline phosphatase (AP) displays sulfatase activity. Such a relationship between these two evolutionarily related enzymes can provide a clearer understanding of past evolutionary pathways and present evolutionary potential. No evidence of a physiological role for AP as a sulfatase was found, but active site features that play a role in the catalysis of both reactions may have been involved in the evolutionary diversification of APs and arylsulfatases.

Human alkyladenine-DNA glycosylase (AAG) catalyzes the excision of a broad range of modified bases, protecting the genome from many types of alkylative and oxidative DNA damage. We have investigated how AAG discriminates against normal DNA bases, while accommodating a structurally diverse set of lesioned bases, by measuring the rates of AAG-catalyzed (kst) and spontaneous N-giycosidic bond hydrolysis (knon) for damaged and undamaged DNA oligonucleotides. The rate enhancements for excision of different bases reveal that AAG is most adept at excising the deaminated lesion hypoxanthine (kst/knon = 108), suggesting that enzymatic activity may have evolved in response to this lesion. Comparisons of the rate enhancements for excision of normal and modified purine nucleobases provide evidence that AAG excludes the normal purines via steric clashes with the exocyclic amino groups of adenine and guanine. However, methylated purines are more chemically labile, and only modest rate enhancements are required for their efficient excision. Base flipping also contributes to specificity as destabilized mismatched base pairs are better substrates than stable WatsonCrick pairs, and many of the lesions recognized by AAG are compromised in their ability to base pair. These findings suggest that AAG reconciles a broad substrate tolerance with the biological imperative to avoid normal DNA by excluding normal bases from the active site rather than by specifically recognizing each lesion. [ABSTRACT FROM AUTHOR]