galactosidase (E. coli)

galactosidase (E. coli)

12 Pages · 2009 · 693 KB · English

1Instititute of Molecular Biology, Howard Hughes Medical Institute and Department of Physics, University of Oregon,. Eugene, Oregon 97403-1229. 2Division of Biochemistry The galactosyl moiety is released to this molecule via a second carbocation-like transition state (the two transition states are

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Direct and indirect roles of His418 in metal binding and in the activity ofbgalactosidase (E coli) Douglas H Juers, 1Beatrice Rob, 2Megan L Dugdale, 2Nastaron Rahimzadeh, 2 Clarence Giang, 2Michelle Lee, 2Brian W Matthews, 1and Reuben E Huber 2* 1Instititute of Molecular Biology, Howard Hughes Medical Institute and Department of Physics, University of Oregon, Eugene, Oregon 974031229 2Division of Biochemistry, Faculty of Science, University of Calgary, Calgary Alberta, Canada, T2N 1N4 Received 2 January 2009; Revised 2 April 2009; Accepted 9 April 2009 DOI: 101002/pro140 Published online 16 April 2009 proteinscienceorg Abstract: The active site of ßgalactosidase (E coli) contains a Mg 21 ion ligated by Glu416, His418 and Glu461 plus three water molecules A Na 1ion binds nearby To better understand the role of the active site Mg 21 and its ligands, His418 was substituted with Asn, Glu and Phe The Asn418 and Glu418 variants could be crystallized and the structures were shown to be very similar to native enzyme The Glu418 variant showed increased mobility of some residues in the active site, which explains why the substitutions at the Mg 21 site also reduce Na 1binding affinity The Phe variant had reduced stability, bound Mg 21 weakly and could not be crystallized All three variants have low catalytic activity due to large decreases in the degalactosylation rate Large decreases in substrate binding affinity were also observed but transition state analogs bound as well or better than to native The results indicate that His418, together with the Mg 21, modulate the central role of Glu461 in binding and as a general acid/base catalyst in the overall catalytic mechanism Glucose binding as an acceptor was also dramatically decreased, indicating that His418 is very important for the formation of allolactose (the natural inducer of thelacoperon) Keywords:bgalactosidase; magnesium; degalactosylation; galactosylation; sodium Introduction bGalactosidase (EC 32123) fromE coliis a tetra meric enzyme that catalyzes hydrolytic and trans galactosidic reactions 1 onb Dgalactopyranosides 2 Substrates initially bind in a ‘‘shallow’’ mode, 3,4 subse quently moving deeper into the active site so that the glycosidic oxygen is close enough to be protonated by Glu461 (general acid catalysis) and the galactosyl anomeric carbon is close enough to contact the nucle ophile, Glu537 A carbocationlike transition state forms that collapses into anagalactosidic bond between the carboxyl of Glu537 and the C1 of galac tose This first step of the reaction, with rate constant k 2(see Fig 1), is called galactosylation (the enzyme becomes galactosylated) Upon glycosidic bond cleavage, the first product normally diffuses away Water or an acceptor with a Abbreviations:Amp, ampicillin; BSA, bovine serum albumin; DMSO, dimethyl sulfoxide; EDTA, ethylene diamine tetraacetic acid; Glc, glucose; IPTG, isopropylthioß Dgalactopyranoside; oNP, onitrophenol; oNPG, onitrophenylßDgalactopyranoside; PETG, phenylethylthioß Dgalactopyranoside; pNP, p nitrophenol; pNPG, pnitrophenylß Dgalactopyranoside; TES, Ntris(hydroxymethyl) methyl2aminoethanesulfonic acid; ToNPG, thioonitrophenylß Dgalactopyranoside Douglas H Juers’s current address is Department of Physics, Whitman College, Walla Walla WA 99362, USA Grant sponsor: NIH; Grant number: GM20066; Grant sponsor: National Science and Engineering Research Council (Canada); Grant number: 4691 *Correspondence to:Reuben E Huber, Division of Biochemistry, Faculty of Science, University of Calgary, Calgary Alberta, Canada T2N 1N4 Email: [email protected] Published by WileyBlackwell VC2009 The Protein Society PROTEIN SCIENCE 2009 VOL 18:1281—12921281 hydroxyl group then enters and is activated by Glu461 via general base catalysis The galactosyl moiety is released to this molecule via a second carbocationlike transition state (the two transition states are thought to be similar) to form free galactose or an adduct hav ing a galactosidic bond with the acceptor The rate constant for this step isk 3if the reaction is with water (called degalactosylation) ork 4if the reaction is with an acceptor (called transgalactosylation) When lactose is the substrate, some of the Glc (first product) remains bound long enough to react as the acceptor, 1 in which case the product is allolactose, the natural in ducer of the lac operon bGalactosidase requires Mg 2þ or Mn 2þ for full catalytic activity, 5–7 but the exact role of this ion in ca talysis is unclear 3,8–10 The active site also includes a monovalent cation (usually either Na þor K þ) impor tant for activity, which directly ligates the galactosyl O 6hydroxyl during catalysis 3,11 The two ion sites are situated a few A apart in the active site, both very near to an interface between two domains of the protein Crystal structure and site directed mutagenesis experiments have shown that His418, along with Glu 416

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