scholarly journals A Catalytic Metal Ion Interacts with the Cleavage Site G·U Wobble in the HDV Ribozyme†

Biochemistry ◽  
2009 ◽  
Vol 48 (7) ◽  
pp. 1498-1507 ◽  
Author(s):  
Jui-Hui Chen ◽  
Bo Gong ◽  
Philip C. Bevilacqua ◽  
Paul R. Carey ◽  
Barbara L. Golden
Keyword(s):  
Cleavage Site ◽  
Metal Ion ◽  
Hdv Ribozyme ◽  
Catalytic Metal

scholarly journals Characterization of the two 5-aminolaevulinic acid binding sites, the A- and P-sites, of 5-aminolaevulinic acid dehydratase from Escherichia coli

1995 ◽  
Vol 305 (1) ◽  
pp. 151-158 ◽  
Author(s):  
P Spencer ◽  
P M Jordan
Keyword(s):  
Schiff Base ◽  
Binding Sites ◽  
Metal Ion ◽  
Substrate Binding ◽  
Aminolaevulinic Acid ◽  
Acid Dehydratase ◽  
Carboxylate Groups ◽  
Catalytic Metal ◽  
A Site ◽  
Aminolaevulinic Acid Dehydratase

Experiments are described in which the individual properties of the two 5-aminolaevulinic acid (ALA) binding sites, the A-site and the P-site, of 5-aminolaevulinic acid dehydratase (ALAD) have been investigated. The ALA binding affinity at the A-site is greatly enhanced (at least 10-fold) on the binding of the catalytic metal ion (bound at the alpha-site). The nature of the catalytic metal ion, Mg2+ or Zn2+, also gave major variations in the substrate Km, P-site affinity for ALA, the effect of potassium and phosphate ions and the pH-dependence of substrate binding. Modification of the P-site by reaction of the enzyme-substrate Schiff base with NaBH4 and analysis of the reduced adduct by electro-spray mass spectrometry indicated a maximum of 1 mol of substrate incorporated/mol of subunit, correlating with a linear loss of enzyme activity. The reduced Schiff-base adduct was used to investigate substrate binding at the A-site by using rate-of-dialysis analysis. The affinity for ALA at the A-site of Mg alpha Zn beta ALAD was found to determine the Km for the reaction and was pH-dependent, with its affinity increasing from 1 mM at pH 6 to 70 microM at pH 8.5. The affinity of ALA at the P-site of Zn alpha An beta ALAD is proposed to limit the Km at pH values above 7, since the measured Kd for ALA at the A-site in 45 microM Tris, pH 8, was well below the observed Km (600 microM) under the same conditions. The amino group of the ALA molecule bound at the P-site was identified as a critical binding component for the A-site, explaining why ALA binding to ALAD is ordered, with the P-site ALA binding first. Structural requirements for ALA binding at the A- and P-sites have been identified: the P-site requires the carbonyl and carboxylate groups, whereas the A-site requires the amino, carbonyl and carboxylate groups of the substrate.

scholarly journals Structural intermediates of a DNA–ligase complex illuminate the role of the catalytic metal ion and mechanism of phosphodiester bond formation

2019 ◽  
Vol 47 (14) ◽  
pp. 7147-7162 ◽  
Author(s):  
Adele Williamson ◽  
Hanna-Kirsti S Leiros
Keyword(s):  
Active Site ◽  
Metal Ion ◽  
Bond Formation ◽  
Strand Break ◽  
Nucleophilic Attack ◽  
Dna Ligase ◽  
Phosphodiester Bond ◽  
Dna Ligases ◽  
Catalytic Metal ◽  
Ternary Structure

Abstract DNA ligases join adjacent 5′ phosphate (5′P) and 3′ hydroxyl (3′OH) termini of double-stranded DNA via a three-step mechanism requiring a nucleotide cofactor and divalent metal ion. Although considerable structural detail is available for the first two steps, less is known about step 3 where the DNA-backbone is joined or about the cation role at this step. We have captured high-resolution structures of an adenosine triphosphate (ATP)-dependent DNA ligase from Prochlorococcus marinus including a Mn-bound pre-ternary ligase–DNA complex poised for phosphodiester bond formation, and a post-ternary intermediate retaining product DNA and partially occupied AMP in the active site. The pre-ternary structure unambiguously identifies the binding site of the catalytic metal ion and confirms both its role in activating the 3′OH terminus for nucleophilic attack on the 5′P group and stabilizing the pentavalent transition state. The post-ternary structure indicates that DNA distortion and most enzyme-AMP contacts remain after phosphodiester bond formation, implying loss of covalent linkage to the DNA drives release of AMP, rather than active site rearrangement. Additionally, comparisons of this cyanobacterial DNA ligase with homologs from bacteria and bacteriophage pose interesting questions about the structural origin of double-strand break joining activity and the evolution of these ATP-dependent DNA ligase enzymes.

Magnesium Activation of Ribonuclease H. Evidence for One Catalytic Metal Ion

1994 ◽  
Vol 33 (25) ◽  
pp. 5805-5808 ◽  
Author(s):  
C. B. Black ◽  
J. A. Cowan
Keyword(s):  
Metal Ion ◽  
Ribonuclease H ◽  
Catalytic Metal

Catalytic Metal Ion Binding in Enolase: The Crystal Structure of an Enolase-Mn2+-Phosphonoacetohydroxamate Complex at 2.4-.ANG. Resolution,

Biochemistry ◽  
1994 ◽  
Vol 33 (20) ◽  
pp. 6295-6300 ◽  
Author(s):  
Erli Zhang ◽  
Marcos Hatada ◽  
John M. Brewer ◽  
Lukasz Lebioda
Keyword(s):  
Crystal Structure ◽  
Metal Ion ◽  
Ion Binding ◽  
Metal Ion Binding ◽  
Catalytic Metal

scholarly journals Characterization of the Hepatitis C Virus NS2/3 Processing Reaction by Using a Purified Precursor Protein

2001 ◽  
Vol 75 (20) ◽  
pp. 9939-9946 ◽  
Author(s):  
Michele Pallaoro ◽  
Armin Lahm ◽  
Gabriella Biasiol ◽  
Mirko Brunetti ◽  
Caterina Nardella ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Cleavage Site ◽  
Metal Ion ◽  
Precursor Protein ◽  
Site Directed Mutagenesis ◽  
Size Exclusion ◽  
Sequence Alignments ◽  
Processing Rate

ABSTRACT The NS2-NS3 region of the hepatitis C virus polyprotein encodes a proteolytic activity that is required for processing of the NS2/3 junction. Membrane association of NS2 and the autocatalytic nature of the NS2/3 processing event have so far constituted hurdles to the detailed investigation of this reaction. We now report the first biochemical characterization of the self-processing activity of a purified NS2/3 precursor. Using multiple sequence alignments, we were able to define a minimal domain, devoid of membrane-anchoring sequences, which was still capable of performing the processing reaction. This truncated protein was efficiently expressed and processed in Escherichia coli. The processing reaction could be significantly suppressed by growth in minimal medium in the absence of added zinc ions, leading to the accumulation of an unprocessed precursor protein in inclusion bodies. This protein was purified to homogeneity, refolded, and shown to undergo processing at the authentic NS2/NS3 cleavage site with rates comparable to those observed using an in vitro-translated full-length NS2/3 precursor. Size-exclusion chromatography and a dependence of the processing rate on the concentration of truncated NS2/3 suggested a functional multimerization of the precursor protein. However, we were unable to observe trans cleavage activity between cleavage-site mutants and active-site mutants. Furthermore, the cleavage reaction of the wild-type protein was not inhibited by addition of a mutant that was unable to undergo self-processing. Site-directed mutagenesis data and the independence of the processing rate from the nature of the added metal ion argue in favor of NS2/3 being a cysteine protease having Cys993 and His952 as a catalytic dyad. We conclude that a purified protein can efficiently reproduce processing at the NS2/3 site in the absence of additional cofactors.

Sign in / Sign up

Export Citation Format

Share Document