The pH-dependence of the Escherichia coli RNase HII-catalysed Reaction Suggests that an Active Site Carboxylate Group Participates Directly in Catalysis

Bastock, James A.; Webb, Michelle; Grasby, Jane A.

doi:10.1016/j.jmb.2007.01.083

Cited by 14 publications

(14 citation statements)

References 47 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Similar non-linear regression curve fitting problems have been noted previously (32). , the data fit to a simple one-site inhibition model, implying that the value of K I1 is outside the data range (Fig.…”

Section: Inhibition Of Wt T5fen-catalyzed Reactions With Ca 2ϩsupporting

confidence: 78%

Neutralizing Mutations of Carboxylates That Bind Metal 2 in T5 Flap Endonuclease Result in an Enzyme That Still Requires Two Metal Ions

Tomlinson

Syson

Sengerová

et al. 2011

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Flap endonucleases (FENs) are divalent metal ion-dependent phosphodiesterases. Metallonucleases are often assigned a "two-metal ion mechanism" where both metals contact the scissile phosphate diester. The spacing of the two metal ions observed in T5FEN structures appears to preclude this mechanism. However, the overall reaction catalyzed by wild type (WT) T5FEN requires three Mg 2؉ ions, implying that a third ion is needed during catalysis, and so a two-metal ion mechanism remains possible. , essential in all life forms, are members of the 5Ј-nuclease superfamily of structure-specific phosphate diesterases that carry out essential divalent metal ion-dependent nucleic acid hydrolysis (1). FENs act upon 5Ј-bifurcated structures known as flaps formed during lagging strand DNA replication and long patch base excision repair. Hydrolysis predominantly occurs one nucleotide into the 5Ј-duplex adjoining the site of bifurcation. An unusual feature of these enzymes is their high density of active site carboxylates that coordinate essential divalent metal ion cofactors. Bacterial and bacteriophage FENs possess eight active site acidic residues, seven of which are conserved in FENs from higher organisms and also in other members of the 5Ј-nuclease superfamily (1-4). In contrast, a typical metallonuclease active site consists of three or four carboxylates (5). However, a subclass of FEN paralogues, typified by Escherichia coli ExoIX, exists in eubacteria and appear to lack three of the metal-binding carboxylates (6).Although the most common mechanism suggested for metal-dependent phosphoryl transferases involves two metal ions in contact with the scissile phosphate diester, this is not universally accepted (5, 7-10). Debate about the validity or otherwise of two-metal ion catalysis has focused on several enzymes including flap endonucleases (11-16). Crystallographic studies of substrate-free FENs have demonstrated two active site-bound metal ions (14,(17)(18)(19). Metal ion 1 (M1) occupies a similar position in all structures. However, there is some variation in the position of the site coordinating a second metal ion (M2), often observed in FEN crystal structures. The distance between the M1 and M2 sites varies, and the separations observed are generally much greater than the 4 Å required for both metals to contact the same phosphate diester. For example, in bacteriophage T5FEN, 5 the M1-M2 separation of two Mn 2ϩ ions is 8 Å (Fig. 1A) (17). However, in a substrate-free structure of hFEN1, two magnesium ions are bound with a separation of 3.4 Å in one of three proteins bound to proliferating cell nuclear antigen (Fig. 1B) , 3-(N-morpholino)propane-sulfonic acid. 5 In early literature, T5FEN was referred to as T5 5Ј-3Ј-exonuclease and T5 5Ј-nuclease. In early literature, T4FEN was referred to as referred to also as T4 RNase H.

show abstract

Section: Inhibition Of Wt T5fen-catalyzed Reactions With Ca 2ϩsupporting

confidence: 78%

Neutralizing Mutations of Carboxylates That Bind Metal 2 in T5 Flap Endonuclease Result in an Enzyme That Still Requires Two Metal Ions

Tomlinson

Syson

Sengerová

et al. 2011

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

show abstract

“…A possible alternate mechanism is activation of a metal-bound nucleophile by a general base as suggested for EcoRV and RNase H (45,46). However, two ionizations (slope ϭ 2) and pK a values of ϳ6.0 -7.0 are observed in pH profiles of these enzymes (45)(46)(47), which is significantly different from the pH dependence observed with PRORP1. Thus, it is unlikely that PRORP1 uses a general base mechanism for activation of the nucleophile.…”

Section: Ph Dependence Reveals a Single Ionization Important Formentioning

confidence: 85%

Mechanistic Studies Reveal Similar Catalytic Strategies for Phosphodiester Bond Hydrolysis by Protein-only and RNA-dependent Ribonuclease P

Howard¹,

Klemm²,

Fierke

2015

Journal of Biological Chemistry

View full text Add to dashboard Cite

“…4). However, it remains controversial whether an active site carboxyl group directly participates in catalysis as a general base [48].…”

Section: Catalytic Mechanism Of Rnase Hmentioning

confidence: 99%

Ribonuclease H: molecular diversities, substrate binding domains, and catalytic mechanism of the prokaryotic enzymes

2009

View full text Add to dashboard Cite

The prokaryotic genomes, for which complete nucleotide sequences are available, always contain at least one RNase H gene, indicating that RNase H is ubiquitous in all prokaryotic cells. Coupled with its unique substrate specificity, the enzyme has been expected to play crucial roles in the biochemical processes associated with DNA replication, gene expression and DNA repair. The physiological role of prokaryotic RNases H, especially of type 1 RNases H, has been extensively studied using Escherichia coli strains that are defective in RNase HI activity or overproduce RNase HI. However, it is not fully understood yet. By contrast, significant progress has been made in this decade in identifying novel RNases H with respect to their biochemical properties and structures, and elucidating catalytic mechanism and substrate recognition mechanism of RNase H. We review the results of these studies.

show abstract

The pH-dependence of the Escherichia coli RNase HII-catalysed Reaction Suggests that an Active Site Carboxylate Group Participates Directly in Catalysis

Cited by 14 publications

References 47 publications

Neutralizing Mutations of Carboxylates That Bind Metal 2 in T5 Flap Endonuclease Result in an Enzyme That Still Requires Two Metal Ions

Neutralizing Mutations of Carboxylates That Bind Metal 2 in T5 Flap Endonuclease Result in an Enzyme That Still Requires Two Metal Ions

Mechanistic Studies Reveal Similar Catalytic Strategies for Phosphodiester Bond Hydrolysis by Protein-only and RNA-dependent Ribonuclease P

Ribonuclease H: molecular diversities, substrate binding domains, and catalytic mechanism of the prokaryotic enzymes

Contact Info

Product

Resources

About