Enzyme catalysis via control of activation entropy: site-directed mutagenesis of 6,7-Dimethyl-8-ribityllumazine synthase

Fischer, Markus, Haase, Ilka, Kis, Klaus, Meining, Winfried, Ladenstein, Rudolf, Cushman, Mark, Schramek, Nicholas, Huber, Robert and Bacher, Adelbert 2003. Enzyme catalysis via control of activation entropy: site-directed mutagenesis of 6,7-Dimethyl-8-ribityllumazine synthase. Journal of Molecular Biology 326 (3) , pp. 783-793. 10.1016/S0022-2836(02)01473-0

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Abstract

6,7-Dimethyl-8-ribityllumazine synthase (lumazine synthase) catalyses the penultimate step in the biosynthesis of riboflavin. In Bacillus subtilis, 60 lumazine synthase subunits form an icosahedral capsid enclosing a homotrimeric riboflavin synthase unit. The ribH gene specifying the lumazine synthase subunit can be expressed in high yield. All amino acid residues exposed at the surface of the active site cavity were modified by PCR assisted mutagenesis. Polar amino acid residues in direct contact with the enzyme substrates, 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione and 3,4-dihydroxy-2-butanone 4-phosphate, could be replaced with relative impunity with regard to the catalytic properties. Only the replacement of Arg127, which forms a salt bridge with the phosphate group of 3,4-dihydroxy-2-butanone 4-phosphate, reduced the catalytic rate by more than one order of magnitude. Replacement of His88, which is believed to assist in proton transfer reactions, reduced the catalytic activity by about one order of magnitude. Surprisingly, the activation enthalpy of the lumazine synthase reaction exceeds that of the uncatalysed reaction. On the other hand, the free energy of activation of the uncatalysed reaction is characterised by a large entropic term of −37.8 kJ mol−1, whereas the entropy of activation of the enzyme-catalysed reaction is −6.7 kJ mol−1. This suggests that the rate enhancement by the enzyme is predominantly achieved by establishing a favourable topological relation of the two substrates, whereas acid/base catalysis may play a secondary role.

Item Type:	Article
Date Type:	Publication
Status:	Published
Schools:	Biosciences
Publisher:	Elsevier
ISSN:	0022-2836
Last Modified:	24 Jun 2017 11:00
URI:	https://orca.cardiff.ac.uk/id/eprint/70203

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