Abstract
Forty-one aromatic and aliphatic analogs of alpha-ketoglutarate were studied kinetically for their interaction with the alpha-ketoglutarate binding site of gamma-butyrobetaine hydroxylase obtained from Pseudomonas sp. AK1. Together, the compounds represent structural permutations probing the contribution of: 1) the C5 carboxyl group of alpha-ketoglutarate (domain I); 2) the C1-C2 keto acid moiety of alpha-ketoglutarate (domain II); 3) the distance between domains I and II; and 4) the spatial relationship of the two domains required for optimal interaction with the cosubstrate binding site. All compounds were competitive inhibitors for alpha-ketoglutarate (Km 0.018 mM). Functionally, two subsites of the cosubstrate binding site were evident: subsite I for polar interaction with the C5 carboxyl group, and subsite II, comprising of two distinct cis-oriented coordination sites of the catalytic ferrous ion which interact with the C1-C2 keto acid moiety. The most efficient inhibitors were pyridine 2,4-dicarboxylate (Ki 0.0002 mM) and 3,4-dihydroxybenzoate (Ki 0.0006 mM). Both compounds contain a carboxyl group and a chelating moiety corresponding to domains I and II of alpha-ketoglutarate, respectively. The fixed orientation of these groups in both analogs was used to assess intersubsite distance and spatial relationship required for optimal interaction with the cosubstrate binding site. Binding at subsite I and chelation at subsite II were indispensible for effective competitive inhibition. The distance between these two domains also helped determine whether attachment at the cosubstrate binding site would be catalytically productive. This was emphasized by the failure of either oxaloacetate or alpha-ketoadipinate to promote hydroxylation. Optimal interdomain distance, however, was not sufficient for cosubstrate utilization, as pyridine 2,4-dicarboxylate, with an interdomain distance identical to alpha-ketoglutarate in its staggered conformation, did not sustain hydroxylation. In the overall, these studies suggest that alpha-ketoglutarate utilization occurs in a ligand reaction at the active site ferrous ion of gamma-butyrobetaine hydroxylase. This is of particular interest since the delineated stereochemical mode of oxidative decarboxylation could generate the reactive oxo-iron species that was shown experimentally to promote gamma-butyrobetaine hydroxylation by an abstraction-recombination mechanism (Blanchard, J. S., and Englard, S. (1983) Biochemistry 22, 5922-5928; Englard, S., Blanchard, J. S., and Midelfort, C. F. (1985) Biochemistry 24, 1110-1116).
Highlights
Forty-one aromatic and aliphatic analogsof a-keto- eated stereochemical mode of oxidative decarboxylaglutarate were studied kinetically for their interactiotinon could generate the reactive oxo-iron species that with the a-ketoglutarate binding site of y-butyrobe- was shown experimentally to promote 7-butyrobetaine tainehydroxylaseobtainedfromPseudomonas sp. hydroxylation by an abstraction-recombination mech
Two subsites of medium and long chain fatty acids across the inner mitoof the cosubstrate bindingsite were evident: subsiteI chondrial membrane, and is important in thermooefstm(eoufrMrKfdrbaiisc0)ipcn.ii.ttoaeB0ewtln0oiai1to0trt1ihnh2n, ithccnmhsoiotieMbetmmCeir)tsapploa-corornCitufssid2nwoitndhkneg3seer,wteco4ocpof-iatdyntthitrwahailtdoiyyhnidtendiaraceicoCs2icxftd,5aiey4nrrb-bmccrdeooatoinruxccizbesyaioostirlyoa-bx.ongtoye(rrxlTwioKeyughihnl0preaitoc.et0ehuamd0npcoi0d,nos6a--tandaogvi tceexyrTniiyndlehcaslrelitoeusinsodbiganedisnouc(dsteh1wysa-kno3oinetu)mht.tofeCooagtlfiteaectntrychneuapseilctiaaisiprtndehoestrwhxo(mayax3otgyia,fsdeyo4cenma)p-a.ruleenstnsioi,ltdiitzpnohiaennergtitshishceriiytepedmoarftoaepxsrrkhyionaolcabretstliseheosnunitisontnflgin@oathgf-t at chelating moiety corresponding to domainsI and I1 of aIiphaticcarboncenters, eachmediated by aferrous iona-ketoglutarate, respectively
Site would be catalytically productive. This was emy-Rutyrobetaine hydroxylase catalyzes the terminal stepin phasized by the failure of either oxaloacetate or a- carnitine biosynthesis, the hydroxylation of 4-N-trimethyketoadipinate to promote hydroxylation
Summary
Materials-Bovine liver catalase (65,000 units/mg) was obtained from Boehringer Mannheim. a-Keto-[1-"CJglutarate (sodium salt, 59.4 mCi/mmol) was purchased from DuPont-New England Nuclear. The standard 0.7-1111incubation mixtures contained: 15 mM potassium phosphate buffer, pH 7.4, 14 mM sodium ascorbate, 0.2 mM a-ket~-[l-'~C]glutara(t0e.23 pcilpmol), 1mgof catalase, 29 mM y-butyrobetaine, 0.6 mM ferrous ammonium sulfate, and 0.05 pg of the purified enzyme. Linearity of the time course of the reaction up to 20 min under the defined conditions of assay was determined and established for compounds 3, 6, 8, 12-16,18-26, and 31-33 at the highest concentration used in the determination of their respective inhibition constants These were representative of the 41 aliphatic and aromatic compounds listed, with K,values ranging from 0.0002 mM determined for pyridine 2,4-dicarboxylate to 16.9 mM determined for benzene 1,2-dicarboxylate.A t least three different enzyme preparations were used in the course of the present study, and with each nearly identical kinetic constants were obtained as determined for a-ketoglutarate and compounds 12, 14,25, and 26. To study the effect on the overall enzyme reaction of a-ketoglutarate replacement by either a-ketoadipinate, oxaloacetate, or pyridine 2,4-dicarboxylate, formation of [ methyl-'4C]carnitine under standard conditions was determined using [ methyl-'4C]y-butyrobetaine(0.09 pCi/pmol) as substrate. !metl~yl-'~C]Carnitinweas separated from [methyl-'4C]r-butyrobetaineby HPLC, using a Varian model 5500 system equipped with a Varian MicroPak amino acid hydrolysate column
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