Abstract
Recombinant human CuZn superoxide dismutase as expressed in yeast has been crystallized in three different crystal forms. Hexagonal plates grow from 2.4 M ammonium sulfate, pH 7.5, and belong to the space group P6(3)22, with cell dimensions a = b = 113.5(3), c = 151.5(5) A, and Vm = 2.21 A3/dalton for two dimers per asymmetric unit. At 2.0 M ammonium sulfate, pH 7.5, chunky wedges grow in space group C222(1), a = 205.2(6), b = 166.5(4), c = 145.4(4) A with a Vm of 2.43 A3/dalton for eight dimers per asymmetric unit. With polyethylene glycol 8000, pH 7.5-8.0, hexagonal prisms are obtained with cell dimensions a = b = 197.4(6), c = 43.1(2) A, space group P6, and Vm = 2.53 A3/dalton for three dimers per asymmetric unit. All of these forms diffract to high resolution, are stable to x-rays, and appear suitable for determination of the atomic structure. Crystals of the doubly mutated enzyme (Cys6----Ala, Cys111----Ser) grown from both micro- and macroseeds of the wild type protein demonstrate the feasibility of isomorphous crystallization of site-directed mutants of the cloned parent enzyme for comparative structure-function studies.
Highlights
Recombinant human CuZn superoxide dismutase as enzyme
Analysisof the atomic structureof the expressed in yeast has been crystallized in three dif- active site complemented and extended the mechanism proferent crystal forms
Crystals of the doubly mutated enzyme (Cyse + Ala, Cys"' + Ser)grown fromboth micro- and macroseeds of the wild type protein demonstrate the feasibility of isomorphous crystallization remarkably high catalytic rate, due to precollision guidance of the substrate by electrostatic forces (Getzoff et al, 1983; Koppenol, 1982), makes the enzyme a simple model system in which to study therole of electrostatic forces in molecular recognition
Summary
Recombinant human CuZn superoxide dismutase as enzyme. All of these forms diffract to high resolution, are stable to xrays, and appear suitable for determination of the atomic structure. Human CuZn superoxide dismutase is a dimer composed of two identical subunits, each containing 153 amino acids of knownsequence (Barra et al, 1980; Hallewell et al, 1985) together witha Cu(I1) and Zn(I1) (Carrico and Deutsc1h9, 70). The recombinant human enzyme has been expressed at high levels, purified, and characterized from both Escherichia coli of site-directed mutants of the cloned parent enzyme (Hallewell et al, 1985) and yeast.' Both recombinant proteins for comparative structure-function studies. The mechanism and active site structure of CuZn superoxide dismutase alsoofferseveral points of interest.The enzyme's catalytic rate isvery rapid (2 x lo M" s-'; Klug et Crystals were grown by vapordiffusionusing the hanging drop method (McPherson, 1976). The crystalsdescribed above are morphologically similar to themicrocrystals of the 6,6'-dithionicotinic acid-derivatized enzyme described by Jabusch et al (1980)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
