scholarly journals Quantum Mechanical/Molecular Mechanical Study of the HDV Ribozyme: Impact of the Catalytic Metal Ion on the Mechanism

2011 ◽  
Vol 2 (22) ◽  
pp. 2906-2911 ◽  
Author(s):  
Abir Ganguly ◽  
Philip C. Bevilacqua ◽  
Sharon Hammes-Schiffer
Keyword(s):  
Metal Ion ◽  
Quantum Mechanical ◽  
Mechanical Study ◽  
Hdv Ribozyme ◽  
Catalytic Metal

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.

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