Proc. NatL Acad. Sci. USAVol. 79, pp. 3300-3304, May 1982Medical Sciences

Thiamin-responsive maple-syrup-urine disease: Decreased affinityof the mutant branched-chain a-keto acid dehydrogenase fora-ketoisovalerate and thiamin pyrophosphate

(inborn error/vitamin dependency/cofactor binding/multienzyme complex/skin fibroblasts)

DAVID T. CHUANG*, LILY S. Ku, AND RODY P. COXDepartment of Medicine, Cleveland Veterans Administration Medical Center and Case Western Reserve University, Cleveland, Ohio 44106

Communicated by Oscar D. Ratnoff, February 10, 1982

ABSTRACT The biochemical basis for the therapeutic effectsofthiamin in thiamin-responsive maple-syrup-urine disease (MSUD)was investigated in intact and disrupted. fibroblast cultures fromnormals and patients with various forms of MSUD. Decarboxyl-ation of a-keto[1-4C]isovalerate (KIV) by intact cells from a thia-min-responsive MSUD patient was at 30-40% of the normal ratewith or without thiamin in the incubation medium. Under similarconditions, intact classical MSUD fibroblasts failed to decarbox-ylate KIV. Branched-chain a-keto acid (BCKA) dehydrogenaseactivity measured in disrupted cells from the thiamin-responsivesubject showed sigmoidal kinetics in the absence of thiamin py-rophosphate (TPP), with an increased concentration of substrateneeded for half-maximal velocity (KO.5 for KIV = 7 mM vs. 0.05mM in normal cells). When assayed with 0.2 mM TPP present, themutant enzyme showed (i) a shift in kinetics to near Michaelis-Menten type as observed with the normal BCKA dehydrogenaseand (ii) a lower Ko.5 value of 4 mM for KIN, suggesting a TPP-mediated increase in the mutant enzyme's affinity for substrate.By contrast, TPP increased only the Vm. and was without effecton the apparentKm for KIV oftheBCKA dehydrogenase from cellsofnormals and patients with classical MSUD and variant thiamin-nonresponsive MSUD (grade 3). Measurement ofthe apparentKmfor TPP of the BCKA dehydrogenase from thiamin-responsivemutant MSUD cells showed a 16-fold increase in the constant to25 ,IM compared to enzymes from normal or classical MSUDcells. These findings demonstrate that the primary defect in thethiamin-responsive MSUD patient is a reduced affinity of themutant BCKA dehydrogenase for TPP that results in impairedoxidative decarboxylation of BCKA.

Maple-syrup-urine disease (MSUD) is an autosomal recessiveinborn error of metabolism in which the primary defect is thatof oxidative decarboxylation of the branched chain a-keto acids(BCKA) derived from the three branched-chain amino acids:leucine, isoleucine, and valine. MSUD has been classified intoclassical, intermediate, and intermittent types (grades 1, 2, and3) based on the rapidity of onset, severity of disease, and tol-erance for dietary protein (1). Both classical and variant formsof MSUD have a deficiency in BCKA dehydrogenase activity,and the level of residual enzyme activity determines the phe-notype (2). In 1971, Scriver and his colleagues described a fe-male infant with a variant form ofMSUD that responded to thia-min administration at 20 times the normal daily requirement(3). On this regimen, the patient's branched-chain amino acidlevels returned to normal without restriction ofdietary protein.Since the original report, several more patients with thiamin-responsive MSUD have been described (4-6). However, the

biochemical basis of this variant form ofMSUD remained to beestablished.

Recently, our laboratory has developed a reconstituted assaysystem for BCKA dehydrogenase in which disrupted humanfibroblast preparations show 40-60% of the activity seen withintact cells (7). This system minimized variables (e.g., preex-isting substrate pools and membrane effects) to allow a moredirect measurement of enzyme activity. In the disrupted cellassay, normal cells show hyperbolic kinetics with an apparentKm of0.05-0.10 mM for a-ketoisovalerate (KIV), which is sim-ilar to that reported for highly purified bovine BCKA dehydro-genase (8, 9). In contrast, disrupted cells from classical MSUDpatients show altered sigmoidal kinetics with increased con-centrations of the substrate (4-7 mM) needed for half-maximalvelocity (Ko.5). The availability of this sensitive assay permitteda study of thiamin response in normal human fibroblasts andskin fibroblast cultures derived from patients with classicalMSUD, variant grade 3 MSUD, and clinically thiamin-respon-sive MSUD [the patient of Scriver's (WG-34)]. Using the abovesystem, we were able to demonstrate a thiamin pyrophosphate(TPP)-mediated increase in the affinity of BCKA dehydroge-nase for KIV in the thiamin-responsive MSUD. Moreover, themutant enzyme showed an elevated apparent Km value for TPPas compared with the enzyme from the normal or thiamin-non-responsive MSUD patients.

MATERIALS AND METHODSRadioactive Substrates. [1-I4C]Valine (52.9 mCi/mmol; 1 Ci

- 3.7 X 1010 becquerels), [1-'4C]pyruvate (7.5 mCi/mmol),and a-keto[1-_4C]glutarate (53.6 mCi/mmol) were obtainedfrom New England Nuclear. [1-14C]KIV was prepared from the[1-'4C]valine by oxidation with L-amino-acid oxidase (10) andwas purified to >99% purity with a Bio-Rad AG 50W-X8 ion-exchange column.

Cell Strains. Fibroblasts of the thiamin-responsive MSUDvariant, WG-34, originally described by Scriver et aL (3), wereobtained from the Repository for Mutant Human Cell- Strains(Montreal, Canada). Classical MSUD fibroblasts GM-612 andDaMa have been described (7, 11). Grade 3 (thiamin-nonre-sponsive) MSUD variant, ElHa, was provided by J. Steen-John-sen, Oslo, Norway (2, 12).

Cell Culture. Unless otherwise stated, normal and MSUDmutant fibroblasts were grown in monolayer culture in Way-

Abbreviations: MSUD, maple-syrup-urine disease; TPP, thiamin py-rophosphate;. BCKA, branched-chain a-keto acid; KIV,ca-ketoisoval-erate; K0.5, concentration ofsubstrate needed for half-maximal velocity.* To whom reprint requests should be addressed at: Department ofMedicine/Research, Veterans Administration Medical Center, 10701East Blvd., Cleveland, OH 44106.

3300

The piublication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertise-ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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mouth's medium as described (7). To obtain "thiamin-deficient"cells, fibroblasts were grown for two passages in Eagle's mini-mum essential medium (13) without thiamin HCl and with 10%dialyzed fetal calf serum.

Intact Cell Assay. Fibroblasts were harvested by trypsini-zation and washed as described (7). The washed cells (1 x 106in 0.05 ml of Krebs-Ringer phosphate buffer) were suspendedin 0.35 ml of medium containing 0.25 ml of the Krebs buffer(pH 7.4) and a-keto[1-14C]acid with or without 0.005 ml of thia-min HCl (100 mg/ml). Incubations were for 80 min at 35°C with14Co2 collected in a center well containing 0. 1 ml of2 M NaOH.Details of the assay have been described (7). Protein was de-termined by the method of Lowry et al. (14) with bovine serum

albumin as standard.Assay for BCKA Dehydrogenase. The overall reaction cat-

alyzed by the multienzyme complex was assayed by usingfreeze/thaw-disrupted fibroblast suspensions as described (7).The assay mixture contained 50 mM Tris HCl (pH 7.5), 0.2 mMEDTA, 0.35 mM MgCl2, 0.2 mM CoA, 0.2 mM NAD+, 1.4%dialyzed fetal calf serum, the disrupted cell suspension (1 x 106cells with equivalence to 0.3 mg of protein) in 0.05 ml of Krebsbuffer, and [1-14C]KIV (specific radioactivity, 172 cpm/nmol)with or without 0.2 mM TPP in a final volume of 0.37 ml. Theassay mixture with Krebs buffer in place of disrupted cellsserved as a blank. The incubations and remaining procedureswere as described in the intact cell assay.

Assays for Pyruvate and a-Ketoglutarate DehydrogenaseComplexes. Activity ofdichloroacetate-stimulated pyruvate de-hydrogenase complex was assayed by the method of Sheu et al.(15) with modifications. Harvested and washed fibroblasts weretreated with 5 mM dichloroacetate in Krebs buffer as described(15). The cell mixture was then rapidly frozen and stored at-75°C. For assay, the cell suspension was thawed, and 0.2 mgof protein (in 0.05 ml) was added immediately to the assay mix-ture described for BCKA dehydrogenase except that 0.5 mM[1-'4C]pyruvate (specific radioactivity, 1,260 cpm/nmol) was

substituted for [1-4C]KIV. Incubation was for 4 min at 35°C.The remaining steps were identical to those described for BCKAdehydrogenase. The assay for a-ketoglutarate dehydrogenasecomplex was also similar to that for BCKA dehydrogenase ex-

cept that 0.5 mM a-keto[1-14C]glutarate (specific radioactivity,1,100 cpm/nmol) was used as a substrate, and 2.7 mM CaCl2and 2.7 mM MgCl2 were included in the assay mixture. Incu-bation for the latter assay was at 35°C for 10 min.

RESULTS

Decarboxylation of a-Keto Acids by Normal and MSUDFibroblasts. Intact skin fibroblasts from normal, thiamin-re-

sponsive MSUD (WG-34), and classical MSUD (DaMa) subjectsincubated with 3 mM [1-14C]KIV alone showed rates of decar-boxylation of 0. 218 nmol ofCO2 per min/mg of protein for nor-

mal cells and 0.070 nmol ofCO2 per min/mg of protein for thethiamin-responsive variant cells-a rate that is 32% of the nor-

mal rate (Table 1). Intact classical MSUD fibroblasts had no

measurable BCKA decarboxylation under similar conditions.Addition ofsaturating concentrations ofthiamin (4mM) to intactnormal and thiamin-responsive fibroblasts increased activity by44 and 64%, respectively (Table 1). Classical MSUD intact fi-broblasts continued to show no measurable decarboxylation ofKIV in the presence of thiamin. The defect in decarboxylationin thiamin-responsive and classical MSUD fibroblasts was spe-

cific for KIV because pyruvate and a-ketoglutarate were de-carboxylated at normal rates in mutant cells (Table 1). Thiaminstimulated the decarboxylation of pyruvate (20-40%) and a-ke-toglutarate (130-200%).

In the disrupted cell assay, strains of normal skin fibroblastsderived from different subjects showed BCKA dehydrogenaseactivity that ranged from a low of 1.07 to a high of 1.56 nmolof CO2 per min/mg of protein (Table 2). Disrupted cells frompatients with clinically thiamin-responsive MSUD (WG-34) andclassical MSUD (DaMa) had deficient BCKA dehydrogenaseactivity when assayed in the presence of 0.2 mM TPP with 3mM [1-14C]KIV. Fibroblasts from WG-34 had higher residualactivity than did fibroblasts from the classical MSUD patientDaMa. On the other hand, activities of a-ketoglutarate anddichloroacetate-activated pyruvate dehydrogenase complexesappeared to be normal in both MSUD subjects, confirming thespecificity of the defect in MSUD.

Effect ofTPP on BCKA Dehydrogenase. Because TPP is anessential cofactor for BCKA dehydrogenase, the effects of TPPon the enzyme activity of disrupted fibroblasts derived fromnormal subjects and those with classical MSUD (GM-612), vari-ant grade 3 MSUD (ElHa), and the clinically thiamin-respon-sive MSUD (WG-34) were investigated. Disrupted normal cellsexhibited hyperbolic or Michaelis-Menten kinetics over thesubstrate range 0.1-5.0 mM (Fig. 1A). TPP (0.2 mM) increasedonly the Vm. ofthe BCKA dehydrogenase without affecting theKM, which previously had been calculated to be 0.05 mM (7).Disrupted classical-MSUD fibroblasts (GM-612) showed sig-moidal enzyme kinetics with essentially no activity in the 0-1mM range, but activity that approached 30% ofthe normal valueat 5 mM concentration of substrate (Fig. 1B). TPP (0.2 mM)increased the Vm but did not change the K0.5, which was pre-

viously estimated to be 7 mM (7). Fibroblasts from a subjectwith thiamin-nonresponsive grade 3 variant MSUD (ElHa) alsoshowed Michaelis-Menten enzyme kinetics but with an in-

Table 1. Rate of decarboxylation of a-keto acids by intact normal and MSUD fibroblasts

Thiamin-HCl Decarboxylation rate, nmol CO2/min/mg proteinCell line added KIV Pyruvate a-Ketoglutarate

Normal (FG) None 0.218 ± 0.014 0.55 ± 0.01 0.22 ± 0.014 mM 0.314 ± 0.007 0.66 ± 0.01 0.51 ± 0.06

Thiamin-responsive None 0.070 ± 0.006 0.54 ± 0.02 0.17 ± 0.01MSUD (WG-34) 4 mM 0.115 ± 0.006 0.60 ± 0.01 0.52 ± 0.04

Classical MSUD None 0 0.40 ± 0.05 0.39 ± 0.01(DaMa) 4 mM 0 0.57 ± 0.03 0.69 ± 0.03

Cultured fibroblasts were harvested by trypsinization, washed, and suspended in Krebs buffer con-taining the a-keto acid with or without 4 mM thiaminHCl. KIV, pyruvate, and a-ketoglutarate were at3, 0.5, and 5 mM respectively. Specific radioactivities for each 1-1 C-labeled a-keto acid were 172, 1,275,and 270 cpm/nmol, respectively. The incubation was at 350C for 80 min. For each assay, 0.25 mg of protein(1 x 106 cells) was used. The same incubation medium containing an equivalent number of boiled cellswas a blank. Values are means ± SEM (n = 3-5).

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Table 2. Activities of BCKA, pyruvate, and a-ketoglutaratedehydrogenase complexes in disrupted normal and MSUDfibroblasts

Specific activity, nmol C02/ min/mg protein

Cell line BCKA Pyruvate a-Ketoglutarate

NormalPA 0.107 ± 0.007 3.84 ± 0.24 1.56 ± 0.04FR 0.156 ± 0.009 4.68 ± 0.24 2.59 ± 0.15

Thiamin-responsiveMSUD WG-34 0.030 ± 0.002 2.84 ± 0.10 2.27 ± 0.11

Classical MSUDDaMa 0.012 ± 0.002 2.91 ± 0.12 2.32 ± 0.02

Activities ofBCKA, a-ketoglutarate, and dichloroacetate-activated-pyruvate dehydrogenase complexes in freeze/thaw-disrupted fibro-blasts were assayed in the presence of 0.2 mM TPP. The 1- 4C-labeleda-keto acids were at 3 mM (KIV) or 0.5 mM (pyruvate and a-ketoglu-tarate). Specific radioactivities for each substrate were 172, 1,270, and1,142 cpm/nmol, respectively. For each assay, 0.2-0.25 mg of proteinwas added to reaction mixture. Incubation was at 35TC for 80, 4, and10 min for BCKA, pyruvate, and a-ketoglutarate dehydrogenase com-plexes, respectively. Values are means ± SEM (n = 3-5).

creased Km value (2 mM) compared to the normal value (Fig.1C). TPP (0.2 mM) increased the Vm., which was in the normal

0.10 A

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E 0.10 C

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range, but the Km value was again unchanged. In contrast, dis-rupted fibroblasts from the patient with clinically thiamin-re-sponsive MSUD (WG-34), in the absence of added TPP, ex-hibited sigmoidal kinetics with activity strictly dependent onsubstrate concentration (Fig. LD). A Hill plot (Fig. ID Inset)gives a Ko.5 value of7mM and a Hill coefficient of4.0. The Hillcoefficient strongly suggests positive cooperativity of the mu-tant enzyme with the a-keto acid. A saturating concentrationofTPP (0. 2 mM) results in a significant reduction in sigmoidicityto near Michaelis-Menten kinetics (Fig. iD). A Hill plot yieldsa KO5 value of 4 mM and a coefficient of 1.3 in the presenceofTPP (Fig. LD Inset). The Vm. in thiamin-responsive MSUDfibroblasts was determined to be 0.09 nmol/min per mg ofpro-tein with or without 0.2 mM TPP (data not shown), which wassimilar to that measured in normal fibroblasts. These findingsindicate a substantial increase in affinity of the mutant BCKAdehydrogenase for a-keto acid with added TPP.

Effect ofTPP on the Stability ofBCKA Dehydrogenase. Todetermine whether the increase in substrate affinity ofthe thia-min-responsive BCKA dehydrogenase in the presence of TPPis the result ofthe cofactor protecting the multienzyme complexfrom degradation, the time-course of the overall reaction wasstudied. Fig. 2 shows that in the disrupted cell assay of both

0.05 B

0.04

0.03

0.02 -

0.01 U

0.5 1.0 2.0 3.0 4.0 5.0

2.0 3.0 4.0 5.0

FIG. 1. Effects of TPP on the activity of BCKA dehydrogenase in disrupted fibroblasts from normal subjects and patients with various formsof MSUD. Activity of BCKA dehydrogenase in disrupted fibroblasts was measured at various concentrations of KIV in the absence (0, l, o, A) orpresence (@, *, *, A) of 0.2 mM TPP. Specific radioactivity of [1-14C]KIV was 172 cpm/nmol, the incubation was at 35TC for 80 min, and the proteinadded per assay was 0.25 mg (the equivalent of 1 x 106 cells). The activity scale on the ordinate is different for the various phenotypes. (A) Normalsubject Pa (o, *). (B) Classical MSUD patient GM-612 (i, *). (C) Grade 3 variantMSUD patient ElHa (o, *). (D) Thiamin-responsive MSUD patientWG-34 (A, A). (Inset) Hill plots.

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normal and thiamin-responsive mutant fibroblasts (WG-34), thereaction was linear for 80 min, irrespective of the presence ofTPP. Thus the enzyme appeared to be stable during incubationwith or without the cofactor added.

Affinity of BCKA Dehydrogenase for TPP. To further elu-cidate the mechanism by which thiamin produced a therapeuticeffect in the thiamin-responsive patient (WG-34), affinity forTPP of normal and mutant BCKA dehydrogenases was investi-gated. Normal, classical MSUD, and WG-34 mutant cells weregrown in minimal essential medium without thiamin HCl fortwo passages. Cells grown under these conditions showed-30-50% of full BCKA dehydrogenase activity in the absenceofTPP (70 and 50% TPP requirement, respectively). Additionof saturating concentrations of TPP (0.2 mM) restored full ac-tivity. Apparent Km for TPP was obtained from double-recip-rocal plots of the increase in BCKA dehydrogenase activity vs.the concentration of TPP. Fig. 3 shows that BCKA dehydro-genase from both normal and classical MSUD had an apparentKm value of 1.6 AM for TPP; however, the enzyme preparationfrom the thiamin-responsive mutant showed a 16-fold increasein this constant to a concentration of25 AM. These results sug-gest a decrease in the affinity ofBCKA dehydrogenase for TPPin thiamin-responsive MSUD.

Affinity of Dichloroacetate-Activated Pyruvate Dehydro-genase Complex for TPP. To further determine whether theapparent alteration in Km for TPP in thiamin-responsive fibro-blasts is confined to the BCKA dehydrogenase, the affinity con-stant for another TPP-dependent multienzyme, the pyruvatedehydrogenase complex, was also measured. Maximum acti-vation of the pyruvate dehydrogenase was achieved throughdephosphorylation of the multienzyme complex by incubatingintact fibroblasts with 5 mM dichloroacetate. The pretreatedfibroblasts were then frozen and thawed for assay. Activatedpyruvate dehydrogenase complex showed only 10-30% of fullactivity in the absence of TPP (90 and 70% TPP requirement,respectively). A saturating concentration of TPP restored fullactivity. By using this activated preparation, apparent Km valuesof 0.13 AM for TPP were determined for pyruvate dehydro-genase complex from normal, classical MSUD, and thiamin-re-sponsive subjects (Fig. 4).

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0~~~~~~~~~~

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20 40 60 80Incubation time, min

FIG. 2. Activity of BCKA dehydrogenase in disrupted normal andthiamin-responsive fibroblasts as a function of incubation time. Theincubation was at 35TC with (e, A) or without (o, A) 0.2 mM TPP addedto the assay mixture. In the assay, [1-14C]KIV was used at 2 mM (spe-cific radioactivity, 172 cpm/nmol) for normal (FG) cells (a, e) and at5 mM (specific radioactivity, 330 cpm/nmol) for thiamin-responsive(WG-34) cells (A, A).

2.0

1.8

1.6

1.4

° 1.2x1s° 1.0

,

| 0.8

0.6

0.4

0.2

Km = 1.6/LM 25 ILM 0.2 0.5 1.01/TPP, 1M'1

FIG. 3. Double-reciprocal plots of the increase in BCKA dehydro-genase activity vs. the TPP concentration in disrupted fibroblasts froma normal subject, and from classical and thiamin-responsive MSUDpatients. Activity of BCKA dehydrogenase was measured in disruptednormal and mutant fibroblasts that had been grown for two passagesin thiamin-deficient medium without or with increasing concentra-tions of TPP. The incubation was at 3500 for 80 min. [1-'4C]KIV (spe-cific radioactivity, 350 cpm/nmol) was used at 2 mM for normal subjectFR (o) and for thiamin-responsive MSUD patient WG-34 (A) and at 5mM for classical MSUD patient DaMa (a). V0 and V are enzyme ac-tivities in the absence and presence of TPP, respectively.

DISCUSSIONThe present study describes a probable biochemical basis forthe therapeutic effect of thiamin treatment in the thiamin-re-sponsive MSUD variant originally reported by Scriver et al. (3).We have approached this problem by studying the effects ofthiamin and its active derivative TPP on the decarboxylation ofa-keto acids in intact and disrupted cultured fibroblasts derivedfrom normals and subjects with different types ofMSUD. Mea-surements of '4Co2 production in intact fibroblasts demon-strated a deficiency in the decarboxylation of [1-14C]KIV in thia-min-responsive MSUD (30-40% of the normal rates) with orwithout thiamin in the incubation medium. Thus, the in vivo

1.2 1

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0.8 .-

0.60.4

0

A

I Ia a I IKm = 0.13 /uM 0.1 0.2 0.5 1.0

1/TPP,,uM11.5 2.0

FIG. 4. Double-reciprocal plots of the increase in activity vs. TPPconcentration for the dichloroacetate-activated pyruvate dehydroge-nase complex in disrupted fibroblasts from normal subject FR (a), clas-sical MSUD patient DaMa (a), and thiamin-responsive MSUD patientWG-34 (A). The incubation was at 3500 for 4 min immediately afterthawing the fibroblasts; [1-'4C]pyruvate (specific radioactivity, 1,200cpm/nmol) was at 0.5 mM, and 0.15-0.2 mg of protein was added perassay. V0 and V are enzyme activities in the absence and presence ofTPP, respectively.

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thiamin-responsiveness previously documented cannot be con-vincingly measured by the intact cell assay. Moreover, the fail-ure of MSUD fibroblasts to decarboxylate [1-14C]KIV is not theresult of deficient transport of a-keto acids into mitochondriawhere BCKA dehydrogenase resides because both thiamin-re-sponsive and classical MSUD mutants decarboxylate [1-1 C]pyruvate at normal rates (Table 1). Evidence has been pre-sented that pyruvate and BCKA are transported by the samemonocarboxylate carrier into the mitochondrial matrix (16, 17).

The availability of the disrupted cell assay permitted studiesof substrate affinity of BCKA dehydrogenase in normal andmutant fibroblasts over a wide concentration range ofthe a-ketoacid. By using the disrupted-cell system, normal cells exhibitedMichaelis-Menten kinetics, and the apparent Km of 0.05 mMfor KIV (7) was in the range of highly purified bovine BCKAdehydrogenase (8, 9). By contrast, in the absence of TPP,BCKA dehydrogenase from the thiamin-responsive variantshowed sigmoidal kinetics (Hill coefficient = 4.0) with a K0_5of 7 mM, similar to those observed in classical MSUD fibro-blasts (7). The addition ofa saturating concentration of TPP (0.2mM) resulted in a substantial increase in substrate affinity ofthe mutant enzyme as shown by the near Michaelis-Mentenkinetics (Hill coefficient = 1.3) and a reduction ofthe KO.5valueto 4 mM. The latter kinetic pattern resembles that of a grade3, thiamin-nonresponsive MSUD variant (Fig. 1C). Thus, ther-apeutic effects of thiamin on the thiamin-responsive patient canbe viewed as a TPP-mediated shift in the activity of the mutantBCKA dehydrogenase from that resembling the enzyme activityin classical MSUD to that in a grade 3 MSUD, which is lesssubstrate dependent. This probably results in significant oxi-dative decarboxylation of BCKA at low substrate concentrationin vivo, thereby avoiding accumulation of the a-keto acids. Incontrast, TPP is without effect on the apparent Km or K0.5 val-ues for KIV ofBCKA dehydrogenase in normal, grade 3 variant,and classical MSUD fibroblasts (Fig. 1).The above mechanism for thiamin response differs from that

suggested earlier by Danner et al. (4). They studied anotherpatient with thiamin-responsive MSUD (Georgian) and the onereported here (Canadian) and proposed that increased TPP fromthiamin treatment stabilizes BCKA dehydrogenase by decreas-ing its turnover rate. However, in their studies, both the normaland mutant enzymes were protected to the same degree by 0.2mM TPP during heat inactivation at 50°C. Therefore, it is dif-ficult to reconcile their findings with the specific therapeuticeffects of thiamin observed in these patients. Under our assayconditions, the enzyme activity of normal and thiamin-respon-sive fibroblasts was linear during the 80-min incubation irre-spective of the presence of 0.2 mM TPP, supporting the con-tention that the TPP-mediated increase in substrate affinity isnot related to stabilization of the enzyme in the in vitro assay.

Thiamin dependency in thiamin-responsive MSUD is ex-plained by the 16-fold increase in apparent Km for TPP of thismutant BCKA dehydrogenase. The defect in TPP binding ap-pears to be specific for thiamin-responsive MSUD because itis not observed in classical MSUD nor is it present in the otherTPP-dependent enzyme of the mutants, i.e., the pyruvate de-hydrogenase complex. Our findings again differ from those ofDanner et aL (4), who found the same apparent Km for TPP (1.6,uM) of BCKA dehydrogenase in normal and thiamin-respon-sive MSUD fibroblasts. A major difference between the pre-vious and present studies is that we determined the apparentKm value for TPP using fibroblast cultures grown for two pas-sages in medium without thiamin-HCl. Under such conditions,BCKA dehydrogenase showed an 50-70% TPP requirementfor full activity. Lipson et aL (18) have shown a decreased affin-

ity of cystathionine j3-synthase for pyridoxal 5'-phosphate inpyridoxine-responsive homocystinuria by using fibroblasts cul-tured in pyridoxine-depleted medium. The latter defect couldnot be readily demonstrated in pyridoxine-responsive fibro-blasts grown in medium containing a high concentration of theB-6 vitamin.The apparent defect in TPP-binding ofBCKA dehydrogenase

in thiamine-responsive MSUD may be analogous to that ob-served in one type oftransketolase deficiency. Blass and Gibson(19) have reported that fibroblasts from four patients with Wer-nicke-Korsakoff syndrome have a transketolase that has 9- to18-fold higher KO.5 for TPP than does the enzyme from normalfibroblasts. Thiamin dependency also was observed in a patientwith congenital lacticacidemia due to pyruvate dehydrogenasedeficiency (20). Although only a few examples of TPP-depen-dent inborn errors have been reported, it seems probable thatother defects in binding of TPP to dependent enzymes will befound.

In summary, the data presented here demonstrate a TPP-mediated increase in affinity of BCKA dehydrogenase for thea-keto acid that is specific for thiamin-responsive MSUD.Moreover, a defect in the binding of TPP has been implicatedby an elevated apparent Km for TPP for the mutant enzyme.The molecular mechanism that leads to the increase in substrateaffinity is not clear. It is possible that a TPP-mediated confor-mational change in the mutant enzyme renders the a-keto acid-binding site more accessible for the substrate. Further studiesare required to substantiate this possibility.

We thank Ms. Yoshie Hervey for her skillful assistance in cell culture.This investigation was supported by National Institutes of Health GrantAM 26758.

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2. Dancis, J., Hutzler, J., Snyderman, S. E. & Cox, R. P. (1972)J.Pediatr. 81, 312-320.

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Proc. Nad Acad. Sci. USA 79 (1982)

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