scholarly journals Exploring the Role of the Third Active Site Metal Ion in DNA Polymerase η with QM/MM Free Energy Simulations

2018 ◽  
Vol 140 (28) ◽  
pp. 8965-8969 ◽  
Author(s):  
David R. Stevens ◽  
Sharon Hammes-Schiffer
Keyword(s):  
Free Energy ◽  
Dna Polymerase ◽  
Active Site ◽  
Metal Ion ◽  
The Third ◽  
Energy Simulations

scholarly journals Role of the Active Site Guanine in theglmSRibozyme Self-Cleavage Mechanism: Quantum Mechanical/Molecular Mechanical Free Energy Simulations

2015 ◽  
Vol 137 (2) ◽  
pp. 784-798 ◽  
Author(s):  
Sixue Zhang ◽  
Abir Ganguly ◽  
Puja Goyal ◽  
Jamie L. Bingaman ◽  
Philip C. Bevilacqua ◽  
...  
Keyword(s):  
Free Energy ◽  
Active Site ◽  
Quantum Mechanical ◽  
Energy Simulations

scholarly journals The Loop of the TPR1 Subdomain of Phi29 DNA Polymerase Plays a Pivotal Role in Primer-Terminus Stabilization at the Polymerization Active Site

Biomolecules ◽  
2019 ◽  
Vol 9 (11) ◽  
pp. 648
Author(s):  
del Prado ◽  
Santos ◽  
Lázaro ◽  
Salas ◽  
de Vega
Keyword(s):  
Dna Polymerase ◽  
Active Site ◽  
Structural Changes ◽  
Binding Capacity ◽  
Dna Polymerases ◽  
Crystallographic Structure ◽  
Genome Replication ◽  
Terminal Protein ◽  
Phi29 Dna Polymerase

Bacteriophage Phi29 DNA polymerase belongs to the protein-primed subgroup of family B DNA polymerases that use a terminal protein (TP) as a primer to initiate genome replication. The resolution of the crystallographic structure showed that it consists of an N-terminal domain with the exonuclease activity and a C-terminal polymerization domain. It also has two subdomains specific of the protein-primed DNA polymerases; the TP Regions 1 (TPR1) that interacts with TP and DNA, and 2 (TPR2), that couples both processivity and strand displacement to the enzyme. The superimposition of the structures of the apo polymerase and the polymerase in the polymerase/TP heterodimer shows that the structural changes are restricted almost to the TPR1 loop (residues 304–314). In order to study the role of this loop in binding the DNA and the TP, we changed the residues Arg306, Arg308, Phe309, Tyr310, and Lys311 into alanine, and also made the deletion mutant Δ6 lacking residues Arg306–Lys311. The results show a defective TP binding capacity in mutants R306A, F309A, Y310A, and Δ6. The additional impaired primer-terminus stabilization at the polymerization active site in mutants Y310A and Δ6 allows us to propose a role for the Phi29 DNA polymerase TPR1 loop in the proper positioning of the DNA and TP-priming 3’-OH termini at the preinsertion site of the polymerase to enable efficient initiation and further elongation steps during Phi29 TP-DNA replication.

Free Energy Simulations of Active-Site Mutants of Dihydrofolate Reductase

2014 ◽  
Vol 119 (3) ◽  
pp. 906-916 ◽  
Author(s):  
Dvir Doron ◽  
Vanja Stojković ◽  
Lokesh Gakhar ◽  
Alexandra Vardi-Kilshtain ◽  
Amnon Kohen ◽  
...  
Keyword(s):  
Free Energy ◽  
Dihydrofolate Reductase ◽  
Active Site ◽  
Energy Simulations ◽  
Active Site Mutants

scholarly journals Re-examination of the roles of PEP and Mg2+ in the reaction catalysed by the phosphorylated and non-phosphorylated forms of phosphoenolpyruvate carboxylase from leaves of Zea mays

1998 ◽  
Vol 332 (3) ◽  
pp. 633-642 ◽  
Author(s):  
Alejandro TOVAR-MÉNDEZ ◽  
Rogelio RODRÍGUEZ-SOTRES ◽  
Dulce M. LÓPEZ-VALENTÍN ◽  
Rosario A. MUÑOZ-CLARES
Keyword(s):  
Active Site ◽  
Phosphoenolpyruvate Carboxylase ◽  
Metal Ion ◽  
Dissociation Constants ◽  
Physiological Role ◽  
Free Enzyme ◽  
Kinetic Properties ◽  
Allosteric Site ◽  
Physiological Concentration

To study the effects of phosphoenolpyruvate (PEP) and Mg2+ on the activity of the non-phosphorylated and phosphorylated forms of phosphoenolpyruvate carboxylase (PEPC) from Zea maysleaves, steady-state measurements have been carried out with the free forms of PEP (fPEP) and Mg2+ (fMg2+), both in a near-physiological concentration range. At pH 7.3, in the absence of activators, the initial velocity data obtained with both forms of the enzyme are consistent with the exclusive binding of MgPEP to the active site and of fPEP to an activating allosteric site. At pH 8.3, and in the presence of saturating concentrations of glucose 6-phosphate (Glc6P) or Gly, the free species also combined with the active site in the free enzyme, but with dissociation constants at least 35-fold that estimated for MgPEP. The latter dissociation constant was lowered to the same extent by saturating Glc6P and Gly, to approx. one-tenth and one-sixteenth in the non-phosphorylated and phosphorylated enzymes respectively. When Glc6P is present, fPEP binds to the active site in the free enzyme better than fMg2+, whereas the metal ion binds better in the presence of Gly. Saturation of the enzyme with Glc6P abolished the activation by fPEP, consistent with a common binding site, whereas saturation with Gly increased the affinity of the allosteric site for fPEP. Under all the conditions tested, our results suggest that fPEP is not able to combine with the allosteric site in the free enzyme, i.e. it cannot combine until after MgPEP, fPEP or fMg2+ are bound at the active site. The physiological role of Mg2+ in the regulation of the enzyme is only that of a substrate, mainly as part of the MgPEP complex. The kinetic properties of maize leaf PEPC reported here are consistent with the enzyme being well below saturation under the physiological concentrations of fMg2+ and PEP, particularly during the dark period; it is therefore suggested that the basal PEPC activity in vivois very low, but highly responsive to even small changes in the intracellular concentration of its substrate and effectors.

scholarly journals Conversion of 19-oxo[2β-2H]androgens into oestrogens by human placental aromatase. An unexpected stereochemical outcome

1990 ◽  
Vol 268 (3) ◽  
pp. 553-561 ◽  
Author(s):  
P A Cole ◽  
C H Robinson
Keyword(s):  
Active Site ◽  
Extended Period ◽  
Direct Role ◽  
Methyl Group ◽  
Enzyme Active Site ◽  
Model Studies ◽  
The Third ◽  
Intensive Investigation ◽  
Cytochrome P 450

Aromatase is a cytochrome P-450 enzyme that catalyzes the conversion of androgens into oestrogens via sequential oxidations at the 19-methyl group. Despite intensive investigation, the mechanism of the third step, conversion of the 19-aldehydes into oestrogens, has remained unsolved. We have previously found that a pre-enolized 19-al derivative undergoes smooth aromatization in non-enzymic model studies, but the role of enolization by the enzyme in transformations of 19-oxoandrogens has not been previously investigated. The compounds 19-oxo[2 beta-2H]testosterone and 19-oxo[2 beta-2H]androstenedione have now been synthesized. Exposure of either of these compounds to microsomal aromatase, in the absence of NADPH, for an extended period led to no significant 2H loss or epimerization at C-2, leaving open the importance of an active-site base. However, in the presence of NADPH there was an unexpected substrate-dependent difference in the stereoselectivity of H loss at C-2 in the enzyme-induced aromatization of 19-oxo[2 beta-2H]-testosterone versus 19-oxo[2 beta-2H]androstenedione. The aromatization results for 17 beta-ol derivative 19-oxo[2 beta-2H]-testosterone correspond to about 1.2:1 2 beta-H/2 alpha-H loss from unlabelled 19-oxotestosterone. In contrast, aromatization results for 19-oxo[2 beta-2H]androstenedione correspond to at least 11:1 2 beta-H/2 alpha-H loss from unlabelled 19-oxoandrostenedione. This substrate-dependent stereoselectivity implies a direct role for an enzyme active-site base in 2-H removal. Furthermore, these results argue against the proposal that 2 beta-hydroxylation is the obligatory third step in aromatase action.

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