Lactate Monooxygenase - The Journal of Biological Chemistry

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Ute Miih8, Charles H. Williams, Jr.80, and Vincent Masseyh. From the .... monooxygenase (Giegel et al., 1990; Ghisla and Massey, 1991): tyrosine 152 was ...
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BIOL~CICAL CHEMISTRY

Vol. 269, No. 11, Issue of March 18,pp. 7994-8000, 1994 Printed in U.S.A.

Lactate Monooxygenase 111. ADDITIVE CONTRIBUTIONS OFACTIVE SITE RESIDUES TO CATALYTIC EFFICIENCY AND STABILIZATION O F AN ANIONIC TRANSITION STATE* (Received for publication, September 28, 1993, and in revised form, December 2, 1993) Ute Miih8, Charles H. Williams, Jr.80, and Vincent Masseyh From the $Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109-0606 and the §Department of Veterans Affairs Medical Center, Ann Arbor, Michigan 48105

Lactate monooxygenase catalyzes the conversion of L-lactate to acetate, C02, and water with incorporation of molecular oxygen.Several amino acidresidues of lactate monooxygenase had been postulated to interact in specific ways with the bound substrate (Giegel, D. A., Williams, C. H., Jr., and Massey, V. (1990) J. Biol. Chem. 265,662645632).Qrosine 44 and arginine 293 were proposed to form a hydrogen bond and a saltbridge to the carboxyl-moiety of lactate. Tyrosine 152 was suggested to form a hydrogen bond to the a-hydroxyl group and could be involvedin stabilizing a transientcarbanionic intermediate of the substrate. The tyrosine residues were replaced with phenylalanines (Y44F, Y152F), and arginine 293 was mutated to a lysine (R293K). In all cases catalysis was significantly decreased; however, the binding affinity for L-lactate did not decrease. Instead, the Kd measured for Y152F was 10-fold lower than that for the wild type enzyme. Theproducts of turnover with Y152F were similar to those with wild type enzyme, with 7 0 4 0 % of the reaction proceeding to form acetate, COB,and H20. The catalytic reactions with both Y44F and R293K were substantially uncoupled, with between 60 and 80%of the catalytic turnover forming pyruvate and HzOz. For all mutant forms the reoxidation of enzyme with oxygen in the absence of pyruvate occurred at a rate similar to that measured for the wild type enzyme. The mostimportant effect of the mutations was in the ability to stabilize the transition state analog oxalate. A linear relationship was found between the rateof reduction of the enzyme flavinand thedissociation constant for the binding of oxalate, demonstrating that many individual residues contribute to thelowering of the energy of the transition state, in addition to specific functions being assignable to some specificresidues.

pyruvate before reacting with oxygen, the reaction products then being pyruvate andHzOz. For wild type lactatemonooxygenase in its reaction withL-lactate, the uncoupled reaction can only be mimicked artificially, e.g. by reducing the enzyme with lactate and waitingfor pyruvate to dissociate before admitting oxygen to the reduced enzyme (Lockridge et al., 1972). However, normal turnover proceeds exclusively via the coupled pathway. Despite extensive structural and mechanistic similarities with glycolate oxidase and flavocytochrome b,, discussed below, lactate monooxygenase is unique in itsability to carry out thisoxidative decarboxylation. Frommechanistic studies a model of the active site had emerged that indicated the participation of a base in catalysis (Walsh et al., 1973; Ghisla and Massey, 1975, 1977) and the stabilization of reduced flavin intermediates by a protein positive charge located near the flavin N(1) - C(2) = 0 locus (Massey and Palmer, 1966; Massey et al., 1969; Mullerand Massey, 1969). Inactivation of lactate monooxygenase from Mycobacterium phlei with phenylglyoxal was found to be due to a modification of an arginine residue (Peters et al., 1981). Yet, little more was known about the amino acid residues that interact with the bound substrate, until the structures of two related proteins, glycolate oxidase (Lindqvist and Branden, 1989) and flavocytochrome bz (Xia and Mathews, 1990; Xia et al., 1987) were solved. For each residue thatwas in a position to interact with the substrate, there is anidentical amino acid residue in the homologous sequence of lactate monooxygenase (Fig. 2). The following interactions werepostulated for lactate monooxygenase (Giegel et al., 1990; Ghisla and Massey, 1991): tyrosine 152 was proposed to form a hydrogen bond with the substrate a-hydroxy group. Since catalysis is initiated by abstraction of a proton from the substrate a-carbon by histidine with tyrosine 152 may 290 (Miih et al., 1994a), the interaction be especially important during thereduction step.The negative charge on the putative substratecarbanion could be stabilized L-Lactate-monooxygenase (EC 1.13.12.4) catalyzes the oxida- by the tyrosine, through hydrogen bonding with the substrate tion of L-lactate to pyruvate.As outlined in Fig. 1,the reduced a-hydroxy group as proposed in Fig. 2. Arginine 293 and tyroflavin reacts with molecular oxygen to generate H202. Innor- sine 44 were presumed to form a salt bridge and a hydrogen mal turnover pyruvate slow is t o leave the active site ( k g