Kinetic analysis of product release and metal ions in a metallonuclease

Abstract

Most nucleases rely on divalent cations as cofactors to catalyze the hydrolysis of nucleic acid phosphodiester bonds. Here both equilibrium and kinetic experiments are used to test recently proposed models regarding the metal ion dependence of product release and the degree of cooperativity between metal ions bound in the active sites of the homodimeric PvuII endonuclease. Equilibrium fluorescence anisotropy studies indicate that product binding is dramatically weakened in the presence of metal ions. Pre steady state kinetics indicate that product release is at least partially rate limiting. Steady state and pre steady state data fit best to models in which metals remain bound to the enzyme after the release of product. Finally, analysis of cooperative and independent binding models for metal ions indicates that single turnover kinetic data are consistent with little to no positive cooperativity between the two metal ions binding each active site. Keywordsmetallonuclease; mechanism; kinetics; product release; metal ions; cooperativity Nucleases are unique among hydrolytic enzymes in their challenge: To catalyze the cleavage of a phosphodiester bond, one of the most stable bonds in biochemistry [1], in a charge repulsive, polyanionic context. Due the high negative charge density and relatively high natural abundance [2], divalent cations like Mg(II) and Ca(II) are natural cofactors for this process. Since the early 1990's many metallonucleases have been crystallized, and a large fraction of these enzyme active sites feature more than one metal ion [3]. These structures have served as the inspiration for mechanisms involving two metal ions. Indeed, given the steps necessary to achieve the hydrolysis of phosphodiester bonds, it makes mechanistic sense to distribute the various steps between metal ions. In most proposed mechanisms, one metal ion is principally involved in nucleophile activation (metal A in Fig. 1), while the other (metal B) is often depicted as contributing to transition state stabilization but also involved in facilitating product release by coordinating a water molecule which donates a proton to the leaving group.With regard to the roles of the metal ions in various mechanistic steps, most of the attention has been devoted to the early steps of substrate binding [4], nucleophile activation [5], and other aspects of the chemical step. Indeed, these have been analyzed in some detail for the restriction enzyme PvuII endonuclease [3,6]. Like many of its relatives, this metallonuclease ; E-mail: cdup@umsl.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal perta...

On the Divalent Metal Ion Dependence of DNA Cleavage by Restriction Endonucleases of the EcoRI Family

“…Recently published single-turnover DNA cleavage data for PvuII as a function of Mg 2+ concentration indicate that for the PvuII system, catalysis involving one Mg 2+ per active site can indeed occur, but that a more efficient twometal-ion mechanism can be operative under higher Mg 2+ concentrations. 75 It was shown in the same study that one Ca 2+ or Mg 2+ per active site stimulates substrate binding similarly, but that a second Ca 2+ dramatically increases DNA binding affinity, while there is only a modest increase for Mg 2+ (see also Bellamy et al 76 ). For the PvuII system, it was concluded that the "more efficient two-metal-ion mechanism can be operative under saturating metal ion (in vitro) conditions".…”

Nucleophile activation in PD…(D/E)xK metallonucleases: An experimental and computational pKa study

“…It is very difficult to clearly interpret these data: While k cat is supposed to represent the chemical step, in reality its value also reflects the rate-limiting step. In the case of many enzymes including metallonucleases, this step is product release [45]. The composition of K m is also complex, comprised of a ratio of rate constants for enzyme-substrate complex formation and breakdown.…”

“…Modeling the nucleic acid using the more appropriate nonlinear form of the Poisson-Boltzmann equation [56, 57] is beyond the scope of this study. However, we have published kinetic evidence to support a mechanism in which the enzyme binds metal ion(s) before substrate [45]. In this scenario, the nucleophile can form in the absence of DNA.…”

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