Identification of a Potential General Acid/Base in the Reversible Phosphoryl Transfer Reactions Catalyzed by Tyrosine Recombinases: Flp H305

Abstract

Flp provides a unique opportunity to apply the tools of chemical biology to phosphoryl transfer reactions. Flp and other tyrosine recombinases catalyze site-specific DNA rearrangements via a phosphotyrosine intermediate. Unlike most related enzymes, Flp's nucleophilic tyrosine derives from a different protomer than the remainder of its active site. Because the tyrosine can be supplied exogenously, nonnatural synthetic analogs can be used. Here we examine the catalytic role of Flp's conserved H305. DNA cleavage was studied using a peptide containing either tyrosine (pKa congruent with 10) or 3-fluoro-tyrosine (pKa congruent with 8.4). Religation was studied using DNA substrates with 3'-phospho-cresol (pKa congruent with 10) or 3'-para-nitro-phenol (pKa congruent with 7.1). In both cases, the tyrosine analog with the lower pKa specifically restored the activity of an H305 mutant. These results provide experimental evidence that this conserved histidine functions as a general acid/base catalyst in tyrosine recombinases.

Structural and Kinetic Analysis of Bacillus subtilis N-Acetylglucosaminidase Reveals a Unique Asp-His Dyad Mechanism

“…Sialytransferases of family 42 glycosyltransferases have been reported to utilize a histidine as the base catalyst that abstracts the proton from the nucleophilic hydroxyl group of the sugar acceptor, thereby facilitating attack on the CMP-Neu5Ac donor nucleotide (49,50). Further precedence for the role of histidine residues as base catalysts are reported (51,52).…”

The Partitioning and Copy Number Control Systems of the Selfish Yeast Plasmid: An Optimized Molecular Design for Stable Persistence in Host Cells

“…In addition, a conserved glutamic/aspartic acid reside appears to contribute structurally to the functional state of the active site (74). In Flp, the pentad residues of Arg-191, Lys-223, His-305, Arg-308 and Trp-330, assisted by Asp-194, serve to balance the negative charge on the scissile phosphate as well as to activate and orient the Tyr-343 nucleophile (75-78) (Figure 8B). The strand cleavage step results in the covalent attachment of the tyrosine to the 3’-phopshate end and the exposure of the 5’-hydroxyl group adjacent to it.…”

Kinetic Isotope Effects for Alkaline Phosphatase Reactions: Implications for the Role of Active-Site Metal Ions in Catalysis

“…The clearest evidence for this phenomenon has been obtained with glycosyl and phosphoryl transfer enzymes that utilize general acid catalysis. [72][73][74][75][76][77] For these enzymes, mutation of the residue that acts as the proton donor has a larger deleterious effect for the reactions of substrates with poor leaving groups -i.e., substrates with high pK a leaving groups are more greatly impeded by the mutation than substrates with low pK a leaving groups. These observations suggest that general acid catalysis makes a larger contribution to catalytic proficiency for substrates with poor leaving groups than for those with good leaving groups, as might be expected based on differential proton affinities.…”