tr()()R(ir.,, . ,r,,nrrsr ny 17. 400-409(1989)

LactateDehydrogenase-Catalyzed Regeneration of NADfrom NADHfor Use in -CatalyzedSynthesis

H. Kr,trH CsENnulr AND Geoncs M. WSTTESTDES

Department of Chemistrt', Huruard Uniuersity, Cambridge, Massachusetts 02138

ReceiuedOctober 24, 1988

Lactate dehydrogenase(LDH, rabbit muscle. EC 1.1.1.27),coupled with pyruvic and gfyoxylic acids as oxidants, regeneratesNAD in .situfor enzyme-catalyzed oxidations. The use of LDH has advantages of convenience, stability of the regenerating system, and econ- omy. Pyruvate and glyoxylate differ in their advantages and disadvantages as oxidants. Pyruvate analogs, monomethyl oxalate and S-methyl thiooxalate, were examined as possi- ble oxidants but were not substrates of . o rg8gAcademicpress, Inc.

INTRODUCTION

We herein describethe use of l-lactate dehydrogenase(LDH.r rabbit muscle, EC Ll.1.27). with pyruvic and gly'ory'licacids as oxidants. to regenerateNAD for enzyme-cataly'zed.preparative oxidations. NAD regenerationusing LDH hasad- vantagesof convenience.stability' of the regeneratingenzy'me and reagents,and econom)'.In sy'stemsnot sensitiveto deactivationby glyoxylate.the high redox potentialof glyoxylate is an advantage. are useful as catalysts for organic synthesis (1), and methods for regenerationhave made synthetic applicationsof even cofactor-requiring enzvmes practical (2). Although severalgood to excellent systemsregenerate the reduced nicotinamide cofactors, NAD(P)H (2, 3), methods for regeneratingthe oxidized cofactors. NAD(P), are less developed and are currently being investi- gated (4-7). A frequent problem of enzyme-catalyzedoxidations is not the co- factor-regeneratingstep but the oxidation of substratein the synthetic step. Bio- chemical oxidations may suffer from unfavorable thermodynamics and noncompetitive and uncompetitive product inhibition (4, 8). LDH catalyzes the completely stereoselectivereduction of pyruvic acid (with concomitantoxidation of NADH) to r,-lacticarcid, Eq. il1, R : CHr e, l0). LDH also acceptsas substratesa variety of other a-oxo acids (l l, l2), including gly- oxylic acid,which is reducedin the presenceof NADH to glycolicacid. Eq. [], R

' Abbreviationsused: FMN, flavin mononucleotide; G6P. glucose 6-phosphate; G6PDH, glucose-6- phosphatedehydrogenase; GluDH, glutamate dehydrogenase; Gly-Gly, glycylglycine; Hepes, 4-(2- hydroxyethyl-l-piperazineethanesulfonic acid; HLADH, horse liver ; aKG, ammonium a-ketoglutarate; LDH, L-lactate dehydrogenase; Mops, 4-morpholinepropanesulfonic acid: TEA. triethanolamine.

0045-2068/89$3.00 Copyright O 1989by Academic Press, Inc. All rights of reproduction in any form reserved. LACTATE DEHYDROGENASE-CAT ALY7.ED REGENERATION 401

: H (1J-15).The utility of LDH in preparativereductions. its easeof immobiliza- tion and manipulation.and its stabilitywhen protectedfrom autoxidationhave been demc-rnstrated(12, I6\.

NADH NAD 8\?'n\/ ul R^coo- LDH n^coo- R= cHs, H

We have examined LDH as a catalyst for NAD regeneration.The two most commonly reported methods for regeneratingNAD(P) use ammonium a-ketoglu- tarate (aKG) and glutamatedehydrogenase (GluDH, EC L4.1.3) (4,8, 17) or FMN/Or. with (6) or without (4, 18,19) FMN reductase(EC 1.6.8.1) as a catalyst. LDH is lessexpensive and higher in specificactivity than both GIuDH and FMN reductuse.rUnlike FMNior. LDH pern-ritsNAI) regenerertionunder anaerobic conditions (many enzymesare deactivatedb1, dior,r-gen. superoxide. and perox- ide). Pyruvic and glyoxylic acids are less erpensive than a-ketoglutaricacid and FMN,3 and glyoxylate(Eo: -0.90 Va)is more stron-elyoxidizing than a-KG (E; - -0.l2l V for reductive amination). Potentialobstacles to the useof pyruvateas the oridant were the nonproductive binding of pyruvate to LDH (20) and the condensationsof pyruvate with itself and NAD (10). Both Mgt* and LDH catalyzethe enolizationof pyruvate and thus the nucleophilicaddition of enol pyruvate to C--1of NAD (21). Becauseglyoxylate cannotenolize. it shouldnot undergocondensations as pyruvatedoes. Also. since glyoxylateexists in aqueoussolution as thc hydrate.it shouldbe lesssoluble in organicsolvents than p1'ruvateand thus more easily'separatedfrom products. A potentialproblem with the use of glyo.rylate.hou'ever, was the NAD-linked oxidationof glyoxylateby LDH (14,22). At high pH (pH 9), LDH oxidizesthe hydrate of glyoxylate as an analog of lactate, Eq . l2l. This reaction nor only consumesNAD but also generatesoxalate, which is a noncompetitiveinhibitor of LDH with respectto pyruvate (and presumablythe oxo form of gly,orl'latet(2J).

NAD NADH g -r4.* ?' !_-/ o L2l lrAcoo- *o^coo- LDH ,-,oAaoo-

RESULTSAND DISCUSSION

Our work beganwith a searchfor substratesof LDH other than pyruvate which would be good oxidizing agentsfor NAD regeneration.We measuredthe kinetic

r Cost and specificactivity of LDH vs GIuDH are $0.71l1000U and 1000U/mg vs $4.7/1000U and 40 U/mg. respectively.FMN reductase(50 U/mg) requiresisolation (Ref. (9)). Prices(Sigma. 1988)are for enzymes as researchbiochemicals and thereforerepresent upper limits. One thousandunits (l U : I pmol/min) is approximately the amount of enzymatic activity required to generate1 mol of product/ day. 3 Prices of pyruvic (957o),glyoxylic (50% wlw aqueous solution), and a-ketoglutaric g5%) acids are 7.6, 3.5, and 15 $/mol, respectively (Aldrich and ICN Biochemicals, 1988). a Reduction potentialsare relative to the half-reaction2H* + 2 e --->H". for which E^: _0.42Y at pH7. 402 CHENAULT AND WHITESIDES

.g E 200 E

100

0 06121824 V/[glyoxylate](mL min -1)

Ftc. L Eadie-Hofstee plot used to determineK^(app) of LDH for glyoxylate in the absence(O) and presence (C) of I mn sodium vanadate.

constants of LDH with glyoxylate (pH 7.0, 25'C) and found the apparent Michaelis constant,5K,,(app), to be 25 mr'a(Fig. 1). This value was in reasonable agreementwith previousdeterminations (13-15) and was significantlyhigher than thatfor pyruvate(K,,:0.15-0.33 mM) ( 13,15,25). The maximalvelocity (y,u*) for the reductionof glyoxylate was approximatelythe sameas that with pyruvate and was at least l0a times faster than the oxidation of gly'oxirlatc(ne detectedno oxidation with 50 mv glyoxylate and 2.8 mr'aNAD). At concentrationsabove -50 mu, glyoxylate inhibited its own reduction by LDH. Since glyoxylate exists in aqueous solution as the hydrate and since vanadate has been shown to catalyzethe hydration/dehydration of glyoxylate (25), we also measuredthe kinetic constants of LDH with glyoxylate in the presenceof I mrra vanadate(Fig. l). The K,,,(app)was reducedto 8 mv. but Vn,,,,also dropped by nearly half. In the presenceof l0 mHrvanadate. LDH lost all activity. We did not pursue the mechanism of this inhibition nor the further use of vanadate as a catalyst in NAD-regeneratingsystems. Monomethyl oxalate (1) and S-methyl thiooxalate (2) were tested as pyruvate analogs,but neither was a substrateof LDH (V < 0.005%of that for pyruvate). o o tl tl ^,, ^A^^^- \/n3\-./ UUU cH.s^coo- 1 2 ScsEuE I

To measure the stability of the pyruvate (glyoxylate)/LDH system, we incu- bated 0.1 mrraNAD and 50 mM pyruvate (glyoxylate) with and without LDH. The activities of the speciesin solution were determinedperiodically by the enzymatic assay.In all tests, the organic acids and enzyme remainedfully active after 120h.

5 The K- measured is an apparent value because it is based on the total concentration of glyoxylate, regardless of the form(s) it exists in and the species bound by the enzyme. The actual K- is probably much smaller since glyoxylate exists in solution almost completely as the hydrate and yet LDH binds the free aldehydic species as the substrate for reduction. LACTATE DEHYDROGENAS E-C AT ALY ZED REGENERAT I ON 403

In the presenceof pyruvate, active NAD decreasedsomewhat: After 120h, in the presence and absence of LDH, 45 and 60Vo,respectively, of the original active NAD remained. With glyoxylate, in the presence and absenceof LDH, 82 and 8970,respectively, of the original active NAD remained after 120 h. LDH, coupled with pyruvic and glyoxylic acids as oxidants, successfullyregen- erated NAD in oxidations of 4 mmol of glucose 6-phosphate (G6P) to 6-phos- phogluconatecatalyzedby glucose-6-phosphatedehydrogenase (G6PDH) Eq. t3l. The G6P/G6PDH system was chosenfor initial demonstrationsbecause the oxida- tion of G6P is thermodynamically favorable (spontaneoushydrolysis of 6-phos- phogluconolactonerenders it irreversible)and not subject to product inhibition by 6-phosphogluconate.6-Phosphogluconate was isolatedas its barium salt (26) in 70-80% yield. Total turnover numbers (TTN)6 (J) for NAD(H) were 930-1070.At the end of the reactions (4-6 h), both enzymes remained fully active, and the nicotinamide cofactor retainedl6-99% of its original activity. In separatestability studies, soluble G6PDH incubated in the presenceof 50 mM pyruvic or (25'C, ambient atmosphere)remained 86 and707o active, respectively, after 95 h. When 50 mr',rG6P was included in the incubation mixture, G6PDH lost no activity after 95 h.

aoPot2- to,*\ .-: Qr- NAD n^coo- bu'oH 1 \/,/ tou /\I lrRDH/ \ 3 l3l n ^nn- n vvv \ \*,"Nft:"*,o

To compare the present method of NAD regenerationto methods using aKG/ GIuDH and FMN/Or. we used pyruvate/LDH to regenerate NAD for the HLADH-catalyzed oxidation of c'is-1,2-bis(hydroxymethyl)cyclohexane(3) to (+)-(lR,65)-cis-8-oxabicyclo[4.3.0]nonan-7-one(4),Eq. [4] (Tablel) (4,27). Both PAN-immobilized (28) and membrane-enclosed (29) LDH regeneratedNAD effi- ciently and economically. Particularly striking was the stability of LDH, which ffoH \-A=,oH ,- 2 n^coo- 2 NAD 3 \/ I ton t4l n I v O -/\ 2 NADH \ tto 2 ,A.oo- \-ry 4

6 TTN : mol of isolated product/mol of catalytic species (enzyme or cofactor) in reaction. 404 CHENAULTAND WHITESIDES

TABI-E I

Comparisc'rnof Methods of RegencratingNAD tlom NADH lbr thc Oritllrtionol'3 to 4 Catalyzeclby HI.ADH

Pvruvate/LDH Glvoxvlate/LDH aKG/GluDH" FMN/O.n

Regenerating system Oxidant (mmol) Pyruvic acid Glyoxylic acid a-Ketoglutaric FMN (20.3) (l7D ( 170 add.ed acid ( l -53) .or contrnuously)'' Enzyme (units) LDH (410) GIUDH (230) Millimoles of NAD 0.12 0.27 1.2 Synthetic system Millimoles of 3 71 69 14 Units of HLADH -520 2-50 80 Reactiontime (days) 1.5 1.5 l.s Product yield (Vc)' 85 U-i liO TTN. NAD 1000 430 l0 Regeneratingenzyme 3.9 x 106 HLADH 1.7x 104 1.9x 104 l.l x l0l (% Recovered activit "v \ NAD 11 60 ' Regeneratingenz-vme l-s0 l0 HLADH II0" ]] $i mol NAD regenerated'1 14 .rI 150

" Data taken from Ref. (4). h Data taken from Ref. (26). ' Based on isolated. distilled product. d Based on the total cost of NAD. oxidant. and regeneratingenzyme (one-time use). ' Glytlrylate cleactivltcdHLADH. t Grinding of the immobilization gel into smallerparticles by the stir bar increaseclthe sLtdirccarea of the gel and thus the apparentactivin' of the immobilizedLDH. . This irctivlrtionol'Fll-,AI)H (pure hlrsctl irs lronhilizctl poritlcr) riir\ r.ctl()(lueihlc (thlectl'iirls). Its origin is unknou'n.

was greater than that of GIuDH (4). That LDH and GIuDH are cytoplasmic and mitochondrialenzymes, respectively. may accountfor this differencein stability. Mitochondrial enzymes tend to require associationwith mitochondrial mem- branesfor stability and are less stablein solutionthan cytoplasmicenzymes. When glyoxylate (initial concentration: 0.23-0.25u) servedas the oxidant in Reaction [4], however, no product formed. Recovered enzymes showed nearly full LDH activity but no HLADH activity. ln subsequentstability studies,gly- oxylate irreversibly deactivated HLADH by a process that was neither first- nor second-orderTwith respect to the enzyme (Fig. 2). Glyoxylate probably causedits deactivation by condensingwith the arginine in the binding domain of HLADH, in analogy to the deactivation of HLADH by 2,3-butadioneand phenylglyoxal (30).

7 HLADH exists as a dimer. LACTATE DEH Y DROG EN AS E-C AT ALY ZED REG E N ERATIO N 405

I U\J

BO .> 60 C) (J40

o\ 20

0 40 t,h

Ftc. 2. Effects of 0 (C), 0.5 (A), 5.0 (f ), and 50 mt'r (O) glyoxylate and -50mv glyoxylate plus 50 mrr.l cyclohexanol (I) on HLADH.

Cyclohexanol (50 mu) slightly improved the stability of HLADH in the presence of 50 mM glyoxylate. A final attempt at using glyoxylate in Reaction [4] in which glyoxylate was added to the reactor (2.5 mmol h-r) also failed. After 4 h, HLADH retained only 40Voof its activity. The advantagesand disadvantagesof using pyruvate and glyoxylate with LDH to regenerate NAD are summarized in Table 2. Pyruvate/LDH is perhaps the simplest, least expensive, and most stable system for regeneratingNAD for reac- tions in which the oxidation step is thermodynamically favorable (or can be made so by removing or reactingfurther the product). With enzvmesthat are not sus- ceptible to deactivation by glyoxylate. glyoxylate offers advantagesof a high reductionpotential and an evenlow'er-cost than pvruvirte.A disirclv'untageol'l-l)H is its inabilityto oxidizeNADPH. and crKG/GluDHren-rains the only enzymatic method capableof direct regenerationof NADP. aKG has a good reduction potential.In some reactions,the zwitterionic product, r,-glutamate,may be easier to separatefrom products than lactic or glycolic acids. Methods basedon LDH and GIuDH are superior to those basedon the reduction of FMN.

TABLE2 Advantagesand Disadvantages of Using Py.rffiTX$,Yoxylic Acids r.,,,ith LDH to Regenerate

Method Advantages Disadvantages

Pyruvate/LDH Inexpensive oxidant Moderate oxidizing potential High specific activity of enzyme Mild deactivationof NAD by pyruvate Inexpensive enzyme Inability to oxidize NADPH Small K^ for pyruvate Compatibility with other enzymes Glvoxvlate/LDH Very inexpensive oxidant Large K^ for glyoxylate High specific activity of enzyme Deactivation of HLADH Inexpensive enzyme (and other enzymes'l) Strong oxidizing potential Inabilitv to oxidize NADPH Compatibility with NAD 406 CHENAULT AND WHITESIDES

EXPERIME,NTAL

Materialsand methods.LDH (rabbitmuscle, ammonium sulfate suspension), G6PDH (Leuconostocmesenteroide.r, ammonium sulfate suspension), HLADH (lyophilizedpowder), and biochemicalsfrom Sigmawere used without further purification.cis-1,2-Bis(hydroxymethyl)cyclohexane (98%), pyruvic acid(98%), and glyoxylic acid (50%w/w aqueoussolution) from Aldrich were usedwithout furtherpurification. Water was doublydistilled, the secondtime througha Corn- ing AG-1bglass still. The autotitratorassembly consisted of a RadiometerPHM82 pH meter, TTT80 titrator, TTA80 titration assembly,ABU80 autoburette, REA160titrograph, and REA270 pH stat unit. rH and rrc NMR spectrawere takenon Bruker AM-300and AM-250spectrometers, using CHCl.r ('H 6 7.26, t3C 6 77.0 in CDCI3)or sodium3-(trimethylsilyt)-l-propanesulfonate (methyl rH 6 0.00,r3C 6 0.00in DzO)as internalreferences. Infrared spectra were takenon a Perkin-Elmer 598 spectrophotometer.Elemental analyses were performedby SpangMicroanalytical Laboratory. Enzymaticassays. All assayswere basedon the spectrophotometricobserva- tion of the appearanceor disappearanceof NADH at 334nm (e : 6.18 mu-r cm-r).For LDH, theassay was initiated by adding0.01 ml of a solutioncontaining 0.01-0.1U of LDH to 3.00ml of 0.1 v triethanolamine(TEA) buffer.pH 7.6. containing2.3mu sodiumpyruvate and 0.23mrnl NADH. Assay'ofLDH with glyoxylateas substratewas performed similarl-'- except in 0.I rt Hepcstlr Mops/ NaOH buffer,pH 7.0,with 50 mlr glyoxylate.For HLADH.0.01 ml containing 0.01-0.1U of HLADH wasadded to l.l0 ml of 0.1 r'aGly-Gly buffer.pH 8.0. containingg.gmu cyclohexanoland 0.50mr'a NAD. Units were expressedas activity with 3, which hasa maximalvelocity that is 80%of that of cyclohexanol (27).For G6PDH, 0.02 ml containing0.01-0.10 U of G6PDHwas added to 2.98ml 0.1rvr potassium phosphate buffer, pH 7.0or 7.6, containing 9.8 mv G6P,6.9 mna NAD, and3 mrurMgCl2. Pyruvate was assayed by adding0.10 ml containingup to 210nmol of pyruvateto 0.96ml of 0.I r'aTEA. pH 1.6.containing 0.33 mlt NADH and2 U of LDH. Glyoxylatewas assayedby' the methodof Bergmeyerand Lang (J1),substituting LDH andNADH for glyoxylatereductase and NADPH, respec- tively. Total NAD(H) was assayedby the methodof Bernofskyand Swan(32), and oxalatewas assayedusing a diagnostickit from Sigma. Monomethyloxalate. Reactionof oxalic acid (10 g) and methanolin carbon tetrachloride(JJ) gave,after distillation, 267o dimethyl oxalate Isubl. 40'C (0.1 Torr); 'H NMR (CDClr)6 3.89(s); r3CNMR (CDC|-.)6 ,53.-5.l-s7.81 and 27% monomethyloxalate: bp 50-55'C(0.1 Torr) Uit. (JJ) bp -50--s2"C(0.2 Torr)1,'H NMR (CDCh)5 3.94(s, 3 H), 10.57(s, I H); r3CNMR (CDClr)6 -54.2,158.2, 195.5. PotassiumS-methyl thiooxalate. Oxalyl chloride(10 mmol) was convertedin four steps(34) to potassiumS-methyl thiooxalate (30%): rH NMR (DrO)E 2.33 (s). Enzymestabilities. Pyruvic or glyoxylicacid (50 mu) andNAD (0.1mu) were incubatedwith and without LDH (l U) in 0.10u TEA buffer,pH 7.6,25'C. Periodicaliquots were assayed for activitiesof the organicacid, NAD, andLDH. G6PDH(2U) wasincubated in 50mv Tris/HClbuffer, pH 7.5,6.3mu MgCl2, LACTATE DEHYDROGENASE-CAT ALYZED REGENERATION 407

25"C, in the presence of 0, 5, 15, and 50 mM pyruvate or glyoxylic acid. G6PDH was similarly incubated with 50 mM organic acid plus 50 mr'aG6P. Periodically, aliquots were assayedfor enzymatic activity. HLADH (0.5 mg) was incubated in 0.1 rraMops/NaOH buffer, pH 7.0, 25"C in the presence of 0.0, 0.5, 5.0, or 50 mrr,lglyoxylate, with and without 50 mv cyclohexanol. HLADH was similarly incubated with 50 mM pyruvate, with and without cyclohexanol. Periodically, aliquots were assayedfor enzymatic activity. Oxidation of G6P using LDH to regenerate ltlAD. Each reaction contained in23 ml of water: 4 mmol of G6P (disodium salt), 4 mmol of pyruvate or glyoxylate, 3 pr.molof NAD,75 p.mol of DTT, and 75 pmol of MgCl2. Soluble LDH (125 U) and G6PDH (200 U) were added to initiate the reaction. Reaction progresswas moni- tored by recording the volume of 1.000 N NaOH delivered by an autotitrator assemblyto maintain constant pH (pH7.6 with pyruvate, pH 7.0 with glyoxylate). Upon completion of the reaction, the mixture was assayedfor G6PDH, LDH, and NAD and oxalate if glyoxylate had been used as the oxidant. BaClr ' 2H2O (6 mmol) and then 8 ml of ethanol were added to the stirred solution. The precipitate was filtered, dried in uac'uo, and assayed for 6-phosphogluconateas described previously (25). Oxidation of cis-l ,2-bis(hydroxymethyl)c-vc'lohexane(3) using LDH to regener- ate llAD. A 3-liter, three-neckedround-bottomed flask with magnetic stirring bar was chargedwith 3 ( 10.5g, 7 | mmol), pyruvic acid ( 12.2ml, 172mmol) dissolved in 150ml of water plus 45 ml of 4 N NaOH, 37.5 ml of 0.1 r'aGly-Gly buffer. pH 8.0, 520 ml of nitrogen-spargedwater, HLADH (290 mg. -520U). and PAN- immobilized (27) LDH (410 U, 100 ml of gel). A little 4 N NaOH was added to adjust the pH to 8.1, and the mixture was spargedwith nitrogen for 30 min to remove dioxygen. A 50-pl aliquot was assayedfor initial pyruvate concentration. NAD 0.12 mmol) was added to the mixture. and 50 pclwas removedto assayfor initial NAD(H) concentration. Hexane (l liter) was layered over the aqueous solution. and the reaction was stirred gently. The progress of the reaction was monitored by assayingfor pyruvate. After 40 h, the reaction was complete. Enzy- matic assayshowed 110and 77% of the originalHLADH and NAD(H) activities, respectively.remaining. The hexane was removed by steel cannulaand concen- trated to a yellow oil. The gel was allowed to settle and most of the aqueouslayer removed by cannula. The gel was centrifuged and the supernatantdecanted. The gel was then washed twice by resuspensionin water, centrifugation, and decant- ing. All aqueous layers were combined and extracted with ether. Ethereal por- tions were combined with the concentratedhexane solution,dried over MgSOa, and evaporatedunder reduced pressure.Distillation of the crude product gave 8.5 g (85%) of (+)-1IR, 65)-cis-8-oxabicyclo[4.3.0]nonan-7-one(4) as a colorlessoil: bp 65-75'C (0.3Torr); a2; + 43.8'(neat);'H NMR (CDCI3)6 l.l -2.6 (m, 10H), 4.15(2 : 1.2and 5.0H2, respectively,.,Ig.,n : 8.8 Hz, I H each);ir 3.90and dd, "Iui. (neat) 1770cmr. Analytical data were in agreementwith the literature (4,27). The LDH gel assayedfor 150% of its original activity. A similar reaction was run using HLADH and LDH enclosedin dialysis mem- brane tubing (29).Yield of distilled product was 88%. Attempted oxidations of 3 using glyoxylate and PAN-immobilized or mem- 408 CHENAULT AND WHITESIDES

brane-enclosedLDH were identicalto the reactionsdescribed above except that glyoxylatereplaced pyruvate. In a final attempt,a neutral solutionof sodium glyoxylate (l .14u) was addedat a rateof 2.2ml h- I to a biphasicsystem contain- ing 10g of 3, 20mrrl triethanolamine, and 0.12 mmol of NAD in 800ml of degassed water.HLADH (500U) and LDH (1500U) wereenclosed in dialysistubing (8 ml total volume)and submergedin the aqueouslayer, which was then overlayered with hexane.

ACKNOWLEDGMENTS

This work hasbeen supported in part by NtH GrantGM 30367and by a grantfrom Firmenich.SA. H.K.C. gratefullyacknowledges support as an NIH TrainingGrant Trainee.1984-8-5 (Grant 5-T32- GM-07598).

REFERENCE,S

l. JoNes, J. B. (1986)Tetrahedron 42,3351-3403. WnrrEsrnrs, G. M., AND WoNc, C.-H. (1985) Angew. Chem.lnt. Ed, Engl.24,6l7-638. (1985)in Enzymesin OrganicSynthesis (Ciba Founda- tionSymposiumlll)(Porter,R.. andClark.S.. Eds.).Pitman. LondonlWHlrEsroes. G. M.. AND WoNc, C.-H. (1983)Aldrichimicu A<'tu 16,21-31. CHIe,{rA..l. (l97tt) Immobilizedtnzymes- Researchand Development. Halsted. New York. 2. CnpNeulr, H. K., SItroN.E. S.. aNo WuttESrDES.G. M. rlgtttl)irr Biotechnologr and Genetic EnginecringReviews (Russcll. (;. tr.. F.d.t.V'o1.6. lntcrccpt. \\'inrhornc. pp lll-170. 3. CnENeur-r,H. K,, RNDWHrresroes. G. M. (l9tl7)Appl. Biothent.IJitttt'thnol.11, 147-191. 4. LEr,, L. G., eNo WsITESIDES,G. M. (198-5)J. Anter. Chent..!rrr'.107.6999-7008. 5. Meros, J. R., nNo WoNc, C.-H. (1986)J. Org. Chem.51,2388-2389. 6. DnuecrHAMMER,D. G., Rroole,, V. W., nNo WoNc, C.-H. (1985)J. Ors. Chem.50, 5387-5389. 7. KovoscHrNSKr, J., ,qNo SrEcxneN, E. (1988)Tetrahedron Lett.29,3299-3300. 8. LEE, L. G., exo WHITESTDES,G. M. (1986)J. Org. Chem.51,25-36. 9. Hor-snoor, J. J., Lrues, A., SrerNou, S. J., eND RossvnN, M. G. (t97-5)in The Enzymes (Boyer, P. D., Ed.), 3rd Ed., Vol. I I , pp. 191-292,Academic Press. New York: ScHwenr, G. W.. RNo WlNtR. A. D. (1963)irrThc Enzymcs(lloycr. P. I).. I-uld1.[{.. and My'rbak.K.. Eds.)]nd Ird.. Vol. 7, pp. l2-5-1611.Academic l)r'ess.Ncr,r York. 10. Evensp,J., eNo KepleN, N. O. (1973)Atlu. En:,t'ntrtl.37,61-133. ll. Mgrsren, A. (1952)J. Biol. Cltem. 197,309-3 17 : (1949)J. Biol. Chem. 184, I ll-129. 12. Krv, M.-J., nNo WsrrESrDES.G. M. (1988)J. Amer. Chem. Soc..110,2959-2964. 13. LeuonnN, G. (1963)Biot'hent. 2.337,449-161. 14. DuNcnN, R. J. S., nNo TrproN. K. F. (1969)Eur. J. Bioc'hem.11,58-61. 15. LeNe , R. S., aNo Dexx,cn, E. E. (1969)Biochemistry 8,2958-2966. 16. HtnscsBEIN,B. L., AND WHtresroes, G. M. (1982)J. Amer. Chem..lor'. 104.,1,1-5ti-.1.169. 17. WoNc, C.-H., AND Mnros, J. R. (1985)/. Org. Chem.50, 1992-1994:Cnnne.,\. G.. Bovann, R., CnenaoNst,P., AND LooI, R. (1984)Biotechnol. Bioeng.26,560--563; Cnnnr-,q. G.. BovenA, R., LoNcuI, R., eND Rrve,, S. (1985)Enz,vme Microb. Technol. V,597-600. 18. JoNes,J. B., eNo TeyLoR, K. E. (1973)J. Chem. Soc'.Chem. Commun., 20-5-2061JoNEs, J. B., eNo Tayron, K. E. (1976)Canad. J. Chem. 54,2969-2973. 19. Use of polymer-boundFMN: MourerNe. F., LeNoeRs,J.-P.. AND CRrcHroN.R.R.(1987) Eur. J. Biochem. lM, 329-336. 20. FenNeNor,z-SnNros, T., LLUrs, C., eNo Bozxt, J. (1979)J. Mol. Catal.5,247-262. 21. Grunr'tN,J. H., AND Cnroole, R. S. (tqZO)Biochemistry 9,1195-1205. 22. RorraeNro,M., AND CeRna, M. (1969)Biochim. Biophys. Acta 177,421*426. LACTATE DEHYDROGENASE-CATALYZED REGENERATION 409

23. Novoe, W. B., WINER,A. D., GLATD,A. J., nNo Scnwr,nr, G. W. (19,58)J. Biot. Chem.234. I 143-l148. 24. Zpwe, V., AND FnoMM, H. J . (1962)J. Biol. Chem. 237,,1668-1675. 25. Cnerc, M. M., BAFF, M., RND Gnessr,,M. J. (l9gg).J. Amer. Chem. Soc..110,3302_3303. 26. woNc, c.-H., AND wurresroes, G. M. (l9sl) J. Amer. chem. soc. 103,4890-4g99. 27. Jerovnc, I. J., GooosRANo, H. B., LoK, K. P., eNo JoNEs,J. B. (1982)J. Amer. Chem. Soc'. 104, 4659-4665. 28. Pollnx, A., BLUMENFELD,H., WAX, M., BRucHN, R. L., AND Wnrrgsroes. G. M. 0980) "/. Amer. Chem. Soc. 102, 6324-6336. 29. BeoNensKt, M. D., Csr,NnuLr, H. K., Srrr,roN,E. S., eNo WHrresrDEs,G. M. fl987) J. Amer. Chem. Soc. 109, 1283-1285. 30. LaNce,L.G., IlI,RroRonu,J.F.,ANDveLLr,E,B.L.(1974)Biochemistr1, 13,436l-4370. 31. BencrraEyER,H. u., nNo LnNc, G. (1984)inMethods of EnzymaticAnalysis (Bergmeyer, H. u., Bergmeyer,J.. and Grassl,M., Eds.),3rd Ed., Vol.6,pp. 624-621.Verl:rg C'hcmie. Weinheim. 32. BenNoFSKy, C., lNo SweN, M. (1973)Anal. Biochem. 53,452-459. 33. McCI-ELLAND,R. A., NoRMRN, R. O. C., aND TuoMes, C. B. (1972)J. Chem. Sot:.perkin Trans. 1, 578-581. 34. Potassium S-methyl thiooxalate: Nre,ur,R, (J., eNo MRrres, R. (1978)Chem. Ber. ll1.,2ll8- 2122:dipotassiumthiooxalate: Stonr, W., eND MerrES, R. (1975)Angev'. Chem. Int. Ect.Enet. 14,436-437; potassiummonophenyl oxalate: Srussr, J., exo AseI-es, R. H. ( 1980)Biochemistm 19,5505-5512.