Proc. Natl, Acad. Sci. USAVol. 80, pp. 1487-1491, March 1983Biochemistry

Site-to-site directed immobilization of enzymes withbis-NAD analogues

(immobilized multi-enzyme system/oriented enzyme complex/alcohol dehydrogenase/lactate dehydrogenase)

MATS-OLLE MXNSSON, NILS SIEGBAHN, AND KLAus MOSBACHPure and Applied Biochemistry, Chemical Center, University of Lund, Post Office Box 740, S-220 07 Lund, Sweden

Communicated by Nathan 0. Kaplan, November 12, 1982

ABSTRACT Lactate dehydrogenase (L-lactate:NAD' oxido-reductase, EC 1.1.1.27) and alcohol dehydrogenase (alcohol:NAD' oxidoreductase, EC 1.1.1.1) have been crosslinked withglutaraldehyde on agarose beads. The crosslinking was performedwhile the two enzymes were spatially arranged with their activesites facing one another with the aid of a bis-NAD analogue. Sub-sequently the bis-NAD analogue was allowed to diffuse out. Byusing a third enzyme, lipoamide dehydrogenase (NADH:lipoamideoxidoreductase, EC 1.6.4.3), which was also coupled to the samebeads and which competes with lactate dehydrogenase for theNADH produced by alcohol dehydrogenase, the effect of site-to-site directed immobilization was studied. It was found that muchmore NADH than was theoretically expected (50% instead of 19%of produced NADH) was oxidized by lactate dehydrogenase, whichindicates that the NADH was preferentially channeled to lactatedehydrogenase due to the juxtapositioned active sites of the twoenzymes.

Several coimmobilized multistep enzyme systems have beendescribed in the literature (1-6). For example, an immobilizedsystem composed of the sequence malate dehydrogenase/ci-trate synthase showed a consistantly higher overall steady-staterate (6). More recently, a four-enzyme sequence (7) and eventhe enzymes of a complete metabolic cycle, the urea cycle, havebeen coimmobilized to supports (8). In the latter case, the im-mobilized cyclic enzyme system again was more efficient thanthe corresponding soluble system.

These effects have been partly attributed to the close prox-imity of the enzymes and partly to the diffusional restrictionsimposed by the Nernst unstirred layer around the enzymes (2,9).

Bifunctional NAD analogues, bis-NAD, have been describedas useful reagents for affecting affinity precipitation of enzymes(10). In this report we describe the use of such bis-NAD ana-logues to obtain an immobilized two-enzyme system in whichthe two different active sites are facing one another. The cou-pling of lactate dehydrogenase (L-lactate:NAD' oxidoreduc-tase, EC 1.1.1.27) to immobilized alcohol dehydrogenase (al-cohol:NAD' oxidoreductase, EC 1.1.1.1) was carried out withthe directing aid of a bifunctional NAD derivative which actedas a template for formation of the two-enzyme complex, beforethe subsequent crosslinking with glutaraldehyde. By such anarrangement, the active sites would be positioned against oneanother, even after removal of the template, and it could be ex-pected that the diffusion of the product of the first enzyme, inthis case NADH, to the active site of the second enzyme wouldbe facilitated due to the closer proximity and proper orientationof the active sites, a situation that normally would not occur withsoluble enzymes or randomly immobilized species.

This investigation was initiated because such systems mightserve as models for enzyme complexes (11) of consecutively op-erating enzymes, which are believed to be of importance in theregulation of metabolism and in the channeling of labile inter-mediates (12).

MATERIALS AND METHODSHorse liver alcohol dehydrogenase (1.9 units/mg of protein) wasobtained from Boehringer (Mannheim, Federal Republic ofGermany). Beef heart lactate dehydrogenase (520 units/mg ofprotein), pig heart lipoamide dehydrogenase (NADH:lipoamideoxidoreductase, EC 1.6.4.3; 136 units/mg of protein), NAD,NADH, pyruvate, and oxalate were purchased from Sigma.Benzyl alcohol, acetaldehyde, and silica plates for TLC werefrom Merck (Darmstadt, Federal Republic of Germany), tresylchloride was from Fluka (Buchs, Switzerland), Sepharose andDEAE-Sephacel were from Pharmacia (Uppsala, Sweden), andbis-NAD II (10) N6-[(6-aminohexyl)carbamoylmethyl]-NAD (13)can be obtained from Sigma. bis-NAD I was synthesized ac-cording to the procedure for bis-NAD II (10) but with hydrazineinstead of adipic acid dihydrazide. bis-NAD III was synthesizedby condensing two N6-[(6-aminohexyl)carbamoylmethyl]-NADmolecules with adipic acid dichloride. The connection with NADis through the exocyclic N of adenine. The progress of the syn-thesis and the purity of the bis-NAD analogues could be fol-lowed by HPLAC on a column of silica-bound boronic acid (14).

H 0 0 H11*N-CH2-C- NH NH -C-CH2-N*

NAD NAD

bis-NAD-I

H 0 0 HII II

* N-CH2-C-NH-NH- (CH2)6-NH- NH - C - CH2- N*NAD NAD

bis-NAD-II

H 0 0 0 0 H

*N-CH2-C-NH-(CH2)6-NH-C-(CH2)4-C-NH-(CH2)6- NH -C- CH2- N*NAD NAD

bis-NAD-III

Immobilization. Experiment A. Sepharose 4B (2 g of moistgel) was activated with tresyl chloride as described (15) and about12 mg of alcohol dehydrogenase dissolved in 4 ml of 0.2 M so-dium phosphate (pH 7.5) was added. The coupling was allowedto proceed for 2 hr at room temperature, after which the re-maining active groups on the Sepharose were quenched for 2 hrat room temperature with 0.25 M Tris (pH 8.0). After the firstimmobilization of alcohol dehydrogenase to Sepharose, the gel

1487

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1488 Biochemistry: Minsson et al.

was washed with 0.5 M NaCl/0.2 M sodium phosphate, pH 7.5,and then equilibrated for 10 min at room temperature with 0.2M sodium phosphate, pH 7.5/0.05 M oxalate/0.01 M pyrazole.Subsequently, about 200 nmol of bis-NAD was added and al-lowed to equilibrate for 10 min in order to form a strong ternarycomplex with the pyrazole and the active site of alcohol de-hydrogenase (Fig. 1, step 1). The gel was subsequently filteredon a glass filter funnel and the excess bis-NAD was removed bywashing with buffer containing pyrazole and oxalate in order tomaintain the ternary complex. The amount of bis-NAD that re-mained affinity-bound to the alcohol dehydrogenase was cal-culated by subtracting the bis-NAD removed during washingfrom that initially added (as determined spectrophotometri-cally).

Lactate dehydrogenase was then added, the amount beingthe same (in nmol) as that of the bis-NAD calculated to be bound(Fig. 1, step 2). The lactate dehydrogenase that did not affinity-bind to the bis-NAD pointing out from the active site of alcoholdehydrogenase was removed by filtration of the Sepharose beads.Finally, glutaraldehyde, the length of which can vary becauseof polymerization (16), was added to a final concentration of0.06%. Coupling (crosslinking) was allowed to proceed for 2.5hr at room temperature (Fig. 1, step 3). All these steps wereperformed in the buffer containing oxalate and pyrazole in orderto maintain the bis-NAD bound as ternary complex.

After the glutaraldehyde crosslinking step, the gel was sus-pended in 0.25 Tris (pH 8.0) overnight at 40C in order to quenchthe unreacted aldehyde groups of glutaraldehyde. The gel wasthen carefully washed (Fig. 1, step 4) in order to remove the bis-

. ., ADH1

...- ADH LDH

n4

3

ADH

2

:ADH LDH

benzylalcohol ethanof

NAD that had been used as template during the immobilizationsteps above. The gelwas washed three times with 0.2 M sodiumphosphate, pH 7.5/0.5 M sodium chloride and then with so-dium phosphate buffer containing 0.1 M isobutyramide, 0.05 Moxamate, and 1 mM NADH. This treatment was carried out be-cause it was expected that NADH, together with isobutyramideand oxamate, should be capable of forming new ternary com-plexes with alcohol dehydrogenase and lactate dehydrogenase,respectively, which should compete with enzyme-bound bis-NAD, thereby removing it from the active sites.

In a control experiment, alcohol dehydrogenase and lactatedehydrogenase were randomly immobilized to tresyl chloride-activated Sepharose beads. In this experiment 5.0 mg of alcoholdehydrogenase and 3.0 mg of lactate dehydrogenase were usedper 2.5 g of moist activated gel.

In experiment A the gel was also incubated (Fig. 1, step 5)with bis-NAD (50 nmol/per g of moist gel) in the presence of50 mM oxalate in order to saturate the lactate dehydrogenaseactive sites with bis-NAD. Excess bis-NAD was removed bywashing with 0.1 M sodium phosphate, pH 7.5/50mM oxalate.The amount of bis-NAD that was not affinity-bound upon rein-cubation was measured (UV absorbance in the filtrate afterwashing), giving an indirect measurement of the amount thatdid affinity-bind.

Experiment B. Alcohol dehydrogenase (2.0 mg) and lipoam-ide dehydrogenase (3.0 mg) were coupled simultaneously to tre-syl chloride-activated Sepharose 4B (2.5 g of moist gel). Theconditions were the same as for coupling to tresyl chloride-ac-tivated Sepharose 4B in experiment A. Lactate dehydrogenasewas then site-to-site immobilized to alcohol dehydrogenase withbis-NAD as template by the same procedure as for site-to-sitecoupling in experiment A (Fig. 2A).

In a control experiment (Fig. 2B), all three enzymes (alcohol,

A

NAD+ ( NADH,/ NAD+

B5

FIG. 1. Preparation of site-to-site directed alcohol dehydrogenase(ADH)-lactate dehydrogenase (LDH) complex (experiment A). Steps:1, affinity binding of bis-NAD to alcohol dehydrogenase immobilizedon Sepharose in the presence of pyrazole; 2, affinity binding of lactatedehydrogenase to bis-NAD in the presence of oxalate; 3, crosslinkingwith glutaraldehyde; 4, removal of pyrazole, oxalate, and bis-NAD bywashing; 5, test for site-to-site immobilization by addition of bis-NADin the presence of oxalate, giving affinity binding to lactate dehydro-genase and activity in a coupled substrate assay for alcohol dehydro-genase. The oligomeric nature of the enzymes has not been taken intoaccount. E2, bis-NAD; 4, pyrazole; *, oxalate.

NAD+NADH,' NAD, 1NADH,/ NAD+,'

N4NAD+

FIG. 2. Schematic representation of the immobilized three-en-zyme system (experiment B). (A) Only alcohol dehydrogenase (ADH)and lipoamide dehydrogenase (LiDH) were coupled directly to tresylchloride-activated Sepharose 4B; lactate dehydrogenase (LDH) wassubsequently coupled with the site-to-site directing aid of bis-NAD. (B)All three enzymes were simultaneously immobilized. The oligomericnature of the enzyme has not been taken into account.

Proc. Nad Acad. Sci. USA 80 (1983)

Proc. Natl. Acad. Sci. USA 80 (1983) 1489

lactate, and lipoamide dehydrogenases) were immobilized ran-domly to Sepharose beads; 1.2, 0.6, and 2.0 mg, respectively,were used per 2.5 g of activated moist gel.Enzyme Assays. All assays with immobilized enzymes (25-

100 mg of moist gel) were carried out in a recirculating systemusing a flow cell (17) mounted in a Hitachi 181 spectrophotome-ter. The total volume in all assays was 15 ml.

In experiment A (and its control), the alcohol dehydrogenaseactivity was determined in a coupled substrate assay using 5mMbenzyl alcohol and 5 mM acetaldehyde in order to regeneratethe coenzyme in the active site.The required coenzyme NAD was derived from half of the

bis-NAD; the other half was affinity-bound to lactate dehydro-genase (Fig. 1, step 5). The activity of alcohol dehydrogenasewas also measured with free NAD (50,uM) in order to obtain themaximal activity. The buffer for activity measurements was 0.2M sodium phosphate, pH 7.5/50 mM oxalate in order to keepthe bis-NAD affinity-bound to lactate dehydrogenase during theassay. The activity was recorded as the formation of benzal-dehyde which has an extinction coefficient of 1,400 cm- M-at 279 nm (18).

In experiment B, the alcohol dehydrogenase activity was de-termined by using 50 mM ethanol and 1 mM NAD and record-ing the NADH production (absorbance at 340 nm). The bufferwas 0.2 M sodium phosphate at pH 7.5. The activities of lactatedehydrogenase and lipoamide dehydrogenase were determinedas follows. Lactate dehydrogenase was measured in 0.2 M so-dium phosphate (pH 7.5) as the decrease in absorbance at 340nm with 0.15 mM NADH and 5 mM pyruvate. Lipoamide de-hydrogenase was measured by following the reduction of fer-ricyanide (0.75 mM) at 420 nm with 0.2 mM NADH as the sec-ond substrate. The extinction coefficient for reduced ferricyanideis 1,040 cm-' M-1.

In the coupled assay for the three-enzyme system in exper-iment B (and its control), the reduction of ferricyanide (0.75 mM)by lipoamide dehydrogenase was measured with and withoutpyruvate present-i.e., with and without competition betweenlactate dehydrogenase and lipoamide dehydrogenase for NADHproduced by alcohol dehydrogenase (scheme 1). The concen-trations of ethanol and NAD for alcohol dehydrogenase assaywere 50 and 1 mM, respectively; when pyruvate was added, itwas at 5 mM. The amount of NADH oxidized by lactate de-hydrogenase upon addition of pyruvate was measured indi-rectly as decrease in lipoamide dehydrogenase activity.

LiDHNAD

Ferricyanide Ferrocyanide

NAD ADH , NADH

Ethanol AcetaldehydeLDH NAD

Pyruvate Lactate

ADH, alcohol dehydrogenase; LDH, lactate dehydrogenase;LiDH, lipoamide dehydrogenase.

Scheme 1

RESULTS

Confirmation of site-to-site immobilization

In experiment A, crosslinked site-to-site directed alcohol de-

hydrogenase/lactate dehydrogenase preparations were ob-tained by using the three different bis-NAD analogues to orientthe active sites. The object of this first phase in the investigationwas to establish whether or not such site-to-site immobilizationcould be accomplished and, subsequently, to study the effectsof utilizing bis-NAD analogues with different lengths of the spacerbetween the two NAD entities.

Site-to-Site Directed Immobilization. When the immobi-lized alcohol dehydrogenase was incubated with bis-NAD III(Fig. 1, step 1), 68 nmol of bis-NAD was bound per g of moistgel. With the assumption that each of the two active sites boundone bis-NAD molecule, this would correspond to 2.7 mg of im-mobilized alcohol dehydrogenase. To this 68 nmol of bis-NAD,68 nmol of lactate dehydrogenase was added (Fig. 1, step 2), ofwhich 30 nmol was bound. The calculations of the amounts ofaffinity-bound bis-NAD and lactate dehydrogenase were basedon UV-absorbance measurements made on the filtrates afterwashing away the excess. The lactate dehydrogenase moleculesadded were not affinity-bound to all the bis-NAD bound to al-cohol dehydrogenase, probably due to steric hindrance of thetwo enzymes. Alcohol dehydrogenase is dimeric, and bindingof more than one tetrameric lactate dehydrogenase per alcoholdehydrogenase molecule is probably sterically unfavorable. Thiswould imply that the enzyme complex is made up of approxi-mately one lactate dehydrogenase molecule and one liver al-cohol dehydrogenase molecule connected by one bis-NAD.

These steps and the crosslinking were performed in the pres-ence of oxalate and pyrazole in order to form strong ternarycomplexes between the enzymes and the bis-NAD and to en-sure that bis-NAD would remain in the active site. Glutaral-dehyde was then used as a crosslinking reagent (Fig. 1, step 3)and, finally, the bis-NAD and the ternary complex-forming agentswere washed away (Fig. 1, step 4).

Second Addition of bis-NAD. Reincubation with bis-NADwas performed in the presence of oxalate, leading to strong ter-nary complex formation with lactate dehydrogenase (Fig. 1, step5). After the excess of bis-NAD was washed away, the active sitesof lactate dehydrogenase were assumed to contain one bis-NADmolecule, one NAD entity affinity-bound in the active site andthe other pointing outward. Because the alcohol dehydrogen-ase-lactate dehydrogenase complex was formed in the presenceof bis-NAD, the NAD entity that was pointing outward fromlactate dehydrogenase should have been able to reach the activesite of alcohol dehydrogenase and thus become available as ac-tive coenzyme.

Alcohol Dehydrogenase Activity of the Complexes. The al-cohol dehydrogenase activities of the crosslinked enzyme com-plex (alcohol dehydrogenase-lactate dehydrogenase) obtainedunder different conditions are given in Table 1. There was someactivity even prior to incubation with bis-NAD. This probablywas due to the presence of some bis-NAD from the precedingcrosslinking treatment that had not been washed away. It maybe that the crosslinking physically entrapped these bis-NADmolecules between the two active sites.

As much as 45% of the alcohol dehydrogenase activity ob-tained in the presence of excess NAD (50 ,tM; Km for solubleenzyme, 17 tkM) could be obtained by incubation with bis-NADIII, which is recycled in the active site of alcohol dehydrogen-ase. The nominal concentration of NAD available for alcoholdehydrogenase was only 0.05 ,uM (concentration of bis-NAD).The sterically available NAD entity of the other bis-NAD an--II

alogues also allowed alcohol dehydrogenase activity but to a lesserextent. Crosslinking performed in the presence of bis-NAD II(Table 1) and of bis-NAD I (data not shown here) gave the sameresults. However, less activity was obtained after readdition ofthe bis-NAD analogues. The general conclusion that can be drawn

Biochemistry: MAnsson et al.

1490 Biochemistry: MAnsson et al.

Table 1. Activity of immobilized alcohol dehydrogenase-lactate dehydrogenase complexAlcohol dehydrogenase activity, ,umol/min per 100 mg moist gel*

Prior to After reincubationConditions reincubation bis-NAD I bis-NAD II bis-NAD III

Crosslinking in No soluble NAD added 0.134 0.321 0.70 1.02presence of NAD (50 pM) added 1.84 2.05 1.96 2.27bis-NAD III Activity ratiot 7.3 15.7 35.7 44.9

Cross-linking in No soluble NAD added 0.190 0.245 0.538 0.411presence of NAD (50 IM) added 1.69 1.38 1.66 1.66bis-NAD II Activity ratio 11.2 17.8 32.4 24.8

Randomly No soluble NAD added 0 0.069 0.058 0.054coupled NAD (50 ,uM) added 1.46 1.35 1.49 1.35

Activity ratio 0 5.1 3.9 4.0

*Alcohol dehydrogenase activity was measured with a coupled substrate assay (18) using either bis-NAD affinity-bound tolactate dehydrogenase or soluble NAD as coenzyme. Total assay volume was 15 ml with 5mM benzyl alcohol and 5mM acetal-dehyde with or without 50 pM NAD, all in 0.2 M sodium phosphate, pH 7.5/50 mM oxalate.

tActivity ratio = (activity with no NAD/activity with 50 uM NAD) x 100.

is that the optimal steric availability of NAD for alcohol dehy-drogenase results with use of those bis-NAD analogues used inthe actual crosslinking of the two enzymes.Random Coimmobilization. In a control to experiment A, al-

cohol dehydrogenase and lactate dehydrogenase were randomlycoimmobilized on tresyl chloride-activated Sepharose 4B. Thispreparation was then incubated with the different bis-NAD an-alogues in the same way as the gels with the site-to-site directedimmobilized enzymes. Much less activity (4-5%) was observedupon readdition of bis-NAD when the enzymes were randomlyimmobilized (Table 1), most likely because the free end of thebis-NAD affinity-bound to lactate dehydrogenase did not reachto alcohol dehydrogenase since the enzyme active sites were toofar apart.The amount of bis-NAD that did affinity-bind to lactate de-

hydrogenase upon readdition of bis-NAD to the two-enzymecomplex of alcohol dehydrogenase and lactate dehydrogenasewas roughly the same for all three bis-NAD analogues, both forsite-to-site directed coimmobilization and for random coim-mobilization. This shows that the lower activity observed uponbis-NAD readdition in the control experiment was not the resultof a lower bis-NAD concentration.

Immobilized three-enzyme system

In order to study the effects of juxtaposition of the active sitesof alcohol dehydrogenase and lactate dehydrogenase, a thirdenzyme, lipoamide dehydrogenase, was incorporated into thesystem as a "reporter enzyme" (experiment B). This was chosenbecause it is able to compete with lactate dehydrogenase forNADH formed by alcohol dehydrogenase. In the first immo-

bilization step, alcohol dehydrogenase and lipoamide dehydro-genase were coimmobilized randomly to tresyl chloride-acti-vated Sepharose 4B; then lactate dehydrogenase was coupled toalcohol dehydrogenase with the site-to-site directing aid of bis-NAD III.When ethanol and NAD were added to such a system, acetal-

dehyde and NADH were formed by alcohol dehydrogenase. TheNADH that was produced could be reoxidized to NAD eitherby lipoamide dehydrogenase or by lactate dehydrogenase (Fig.2). With no pyruvate present (i.e., no lactate dehydrogenaseactivity), the NADH produced by alcohol dehydrogenase couldonly be oxidized by lipoamide dehydrogenase. The activity wasmeasured as the decrease in absorbance at 420 nm due to fer-ricyanide reduction. When pyruvate was added, lactate dehy-drogenase started to compete with lipoamide dehydrogenase forthe produced NADH. The amount of NADH oxidized by lac-tate dehydrogenase and lipoamide dehydrogenase, respec-tively, would be expected to be determined by the relative totalnumbers of enzyme units of the two enzymes. This was also foundto be the case for the randomly coupled three-enzyme system(Fig. 2B) in which the amount of NADH oxidized by lactate de-hydrogenase was roughly the expected one, 0.5% found and 4.5%expected (Table 2). With the same assumption, 19% of formedNADH would be expected to be oxidized by lactate dehydro-genase in the system of the same three enzymes but with lactatedehydrogenase juxtaposed to alcohol dehydrogenase. How-ever, as much as 50% of the formed NADH was oxidized by lac-tate dehydrogenase which indicates that the NADH was. pref-erentially channeled from alcohol dehydrogenase directly tolactate dehydrogenase. The net effect was that much less NADHbecame available for lipoamide dehydrogenase.

Table 2. Enzyme activities in three-enzyme system

Separate enzyme activities, % NADH oxidized in the coupled assay,umol/min 100 mg moist gel By LiDH By LDHADH LiDH LDH Theoretical Found Theoretical Found

Site-to-site coupling 0.018 1.1 0.26 81 50 19 50Random coupling 0.012 2.2 0.10 95.5 99.5 4.5 0.5

Separate enzyme activities (ADH, alcohol dehydrogenase; LiDH, lipoamide dehydrogenase; LDH, lactate dehydrogenase) ofthe immobilized three-enzyme systems were first measured under their V.. conditions. From these activities the theoreticalvalues for activities in the coupled assay were calculated and expressed as the ratio of found V,,. activities of LDH and LiDH,LiDH/(LiDH + LDH) and LDH/(LiDH + LDH), respectively. LDH activity in the coupled assay was measured as the decreasein LiDH activity upon addition of pyruvate.

Proc. Natl. Acad. Sci. USA 80 (1983)

Proc. Nati Acad. Sci. USA 80 (1983) 1491

DISCUSSIONExperiment A. From the-data in Table 1, it becomes apparent

that one end of the bis-NAD analogues tested can interact withalcohol dehydrogenase while the other half of the molecule isaffinity-bound (in a ternary complex) to lactate dehydrogenase.This implies that the overall geometry of the two enzymes rel-ative to one another, obtained upon crosslinking, is retained alsoafter the bis-NAD template has been washed away. The ge-ometry is even retained to the extent that the bis-NAD analoguethat was used in the cross-linking yields the highest activity ofalcohol dehydrogenase, thus indicating the best fit. The resultsfrom a blank experiment (Table 1) in which the two enzymes hadbeen randomly coupled to Sepharose show that the bis-NADanalogues added in this case do not interact to any large extentwith alcohol dehydrogenase under the conditions used. This ismost likely due to the fact that the two enzymes are too far apartor are not properly oriented toward one another due to the ab-sence of the bis-NAD analogue during coupling.Experiment B. The objective of this study was to investigate

whether by such orientation the "product" i.e., NADH-ofalcohol dehydrogenase would preferentially be channeled tolactate dehydrogenase or to a competing enzyme, such as lipo-amide dehydrogenase, bound in vicinity of the enzyme complexon the same Sepharose beads. Interpretation of the results ob-tained was simplified because the Km values for NADH of lac-tate dehydrogenase and lipoamide dehydrogenase are about thesame, 23 AM (19) and 25 ,AM (unpublished data), respectively.The sum of the lactate dehydrogenase and lipoamide dehydro-genase activities present was much greater than the alcohol de-hydrogenase activity, which led to the rapid oxidation, by eitherof the two enzymes, of the NADH produced by alcohol de-hydrogenase. Thus, neither lactate dehydrogenase nor lipoam-ide dehydrogenase was operating under Vm. conditions. Theresults obtained definitelyprove that-preferential channeling ofNADH to lactate dehydrogenase does occur (50% oxidation byLDH instead of the theoretical value of 19%).

Conclusions. With the aid of bis-NAD analogues, or possiblyalso with other bis compounds such as bis inhibitors, crosslinkedenzyme complexes can be obtained in an oriented way. At thesame time, the active sites are protected during coupling withthe aid of such orienting molecules, and the enzymes are broughtinto close and directed.(active sites juxtaposed) proximity. Suchpreparations may serve as valuable model systems for consec-utively operating enzyme systems in which preferred channel-ing of the intermediates occurs, or for bifunctional enzymes liketryptophan synthase (20) with which the product of one subunitreaction, indole, is channeled to the second subunit for reactionwith. serine leading to tryptophan. In a specific homogeneousenzyme immunoassay technique (enzyme channeling immu-noassay), the channeling of glucose 6-phosphate from hexoki-

nase to glucose-6-phosphate dehydrogenase has been used toobtain a more efficient assay system (21). A site-to-site arrange-ment possibly could further improve the assay. Site-to-site im-mobilized enzymes might also be useful for improving the en-zymic regeneration of coenzymes in a system like the onedescribed (22) in which the coenzyme NAD was covalently cou-pled to alcohol dehydrogenase and regenerated with solublelactate dehydrogenase.

We-thank Professor Per-Olof Larsson for valuable advice concerningthe synthesis of the three bis-NAD analogues and the Swedish NaturalScience Research Council (Grant 2616-107) and the National Board forTechnical Development (Grant 80-3595) for their generous financialsupport.

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