Biomedical subjects

L H Conlan

Publications and source records attributed to L H Conlan.

3 recordsLinked to original sources

PubMed2000

A catalytically deficient active site variant of PvuII endonuclease binds Mg(II) ions.

In efforts to understand the mechanisms of many nucleic acid enzymes, the first site-directed mutations are made at conserved acidic active residues. Almost without exception, the low or null activities of the resulting variants are attributed to the importance of the acidic residue(s) to the ligation of required metal ions. Using (25)Mg NMR spectroscopy as a direct probe of metal ion binding and the homodimeric PvuII restriction endonuclease as a model system, this interpretation is examined and clarified. Our results indicate that Mg(II) binds wild-type PvuII endonuclease in the absence of DNA with a K(d,app) of 1.9 mM. Hill analysis yields an n(H) coefficient of 1.4, a value consistent with the binding of more than one Mg(II) ion per monomer active site. Variable pH studies indicate that two ionizable groups are responsible for Mg(II) binding by wild-type PvuII endonuclease near physiological pH. The pK(a,app) for these ionizations is 6.7, a value which is unusual for acidic residues but consistent with data obtained for critical groups in MunI endonuclease and a number of other hydrolases. To assign residues critical to ligating Mg(II), binding measurements were performed on the low activity catalytic site mutants E68A and D58A. As expected, E68A binds Mg(II) ions very weakly (K(d,app) approximately 40 mM), implicating Glu68 as critical to Mg(II) binding. Interestingly, while D58A has only residual specific activity, it retains an affinity for Mg(II) with a K(d,app) of 3.6 mM and exhibits a Hill coefficient of 0.7. Moreover, in this variant, multiple ionizable groups with pK(a,app) of 7.2 are involved in Mg(II) binding, suggesting a shuffling of Mg(II) ligands in the active site. These data indicate that Asp58 is important for the critical positioning of metal ion(s) required for catalysis.

Binding Sites↗

PubMed1999

Quantitative evaluation of metal ion binding to PvuII restriction endonuclease.

Restriction enzymes are important examples of phosphodiester hydrolysis activity and as such have been of increasing interest to structural biologists. Much of the architecture of endonuclease active sites has been derived from X-ray crystallographic studies. These structures implicate conserved active site acidic residues and the scissile bond of the substrate as coordination ligands of required metal ions. Central to the development of restriction enzyme mechanism is our understanding of the role of metal ion binding in the reaction, an important feature of which is identifying the energetic contributions of the enzyme and the substrate to metal ion affinity. To begin to address this issue, isothermal titration calorimetry (ITC) and 19F NMR spectroscopy have been applied to evaluate metal ion binding by the representative PvuII endonuclease in the absence of substrate. In separate experiments, ITC data demonstrate that PvuII endonuclease binds 2.16 Mn(II) ions and 2.05 Ca(II) metal ions in each monomer active site with Kd values of approximately 1 mM. While neither calorimetry nor protein NMR spectroscopy is directly sensitive to Mg(II) binding to the enzyme, Mn(II) competes with Mg(II) for common sites(s) on PvuII endonuclease. Substitution of the conserved active site carboxylate Glu68 with Ala resulted in a loss of affinity for both equivalents of both Ca(II) and Mn(II). Interestingly, the active site mutant D58A retained an affinity for Mn(II) with Kd approximately 2 mM. Mn(II) paramagnetic broadening in 19F spectra of wild-type and mutant 3-fluorotyrosine PvuII endonucleases are consistent with ITC results. Chemical shift analysis of 3-fluorotyrosine mutant enzymes is consistent with a perturbed conformation for D58A. Therefore, free PvuII endonuclease binds metal ions, and metal ion binding can precede DNA binding. Further, while Glu68 is critical to metal ion binding, Asp58 does not appear to be critical to the binding of at least one metal ion and appears to also have a role in structure. These findings provide impetus for exploring the roles of multiple metal ions in the structure and function of this representative endonuclease.

Calcium↗

PubMed1999

Modulating restriction endonuclease activities and specificities using neutral detergents.

It is well known that type II restriction enzyme activities and specificities can be modulated by altering solution conditions. The addition of co-solvents such as dimethyl sulfoxide (DMSO), alcohols and polyols can promote star activity, which is the cleavage of non-cognate sequences. While neutral detergents are often used to control protein aggregation, little is known about the effect of neutral detergents on restriction enzyme activities and specificities. We report here that BamHI, BglI, BglII, EcoRI, EcoRV, HindIII, MluI, PvuII, SalI and XhoI restriction endonucleases are remarkably tolerant of high concentrations of neutral detergents Triton X-100, CHAPS and octyl glucoside. In most cases, lambda DNA cleavage rates were comparable to those observed in the absence of detergent. Indeed, the specific activities of SalI and XhoI were appreciably increased in the presence of Triton X-100. For all enzymes active in the presence of detergents, sequence specificity toward lambda DNA was not compromised. Assays of star cleavage of pUC18 by EcoRI, PvuII and BamHI endonucleases in equimolar concentrations of Triton X-100 and sucrose revealed reduced star activity in the detergent relative to the sucrose co-solvent. Interestingly, under star activity-promoting conditions, PvuII endonuclease displayed greater fidelity in Triton X-100 than in conventional buffer. Taken altogether, these results suggest that in some cases, neutral detergents can be used to manipulate restriction endonuclease reaction rates and specificities.

Bacteriophage lambda↗