Site-To-Site Directed Immobilization of Enzymes with Bis-NAD Analogues

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

Site-to-site directed immobilization of enzymes with bis-NAD analogues (immobilized multi-enzyme system/oriented enzyme complex/alcohol dehydrogenase/lactate dehydrogenase) MATS-OLLE MXNSSON, NILS SIEGBAHN, AND KLAus MOSBACH Pure 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- This investigation was initiated because such systems might reductase, EC 1.1.1.27) and alcohol dehydrogenase (alcohol: serve as models for enzyme complexes (11) of consecutively op- NAD' oxidoreductase, EC 1.1.1.1) have been crosslinked with erating enzymes, which are believed to be of importance in the glutaraldehyde on agarose beads. The crosslinkingwas performed regulation of metabolism and in the channeling of labile inter- while the two enzymes were spatially arranged with their active mediates (12). sites facing one another with the aid of a bis-NAD analogue. Sub- sequently the bis-NAD analogue was allowed to diffuse out. By using a third enzyme, lipoamide dehydrogenase (NADH:lipoamide MATERIALS AND METHODS oxidoreductase, EC 1.6.4.3), which was also coupled to the same Horse liver alcohol dehydrogenase (1.9 units/mg of protein) was beads and which competes with lactate dehydrogenase for the obtained from Boehringer (Mannheim, Federal Republic of NADH produced by alcohol dehydrogenase, the effect of site-to- Germany). Beef heart lactate dehydrogenase (520 units/mg of site directed immobilization was studied. It was found that much protein), pigheartlipoamide dehydrogenase (NADH:lipoamide more NADH than was theoretically expected (50% instead of 19% oxidoreductase, EC 1.6.4.3; 136 units/mg of protein), NAD, of produced NADH) was oxidized by lactate dehydrogenase, which NADH, pyruvate, and oxalate were purchased from Sigma. indicates that the NADH was preferentially channeled to lactate Benzyl alcohol, acetaldehyde, and silica plates for TLC were dehydrogenase due to the juxtapositioned active sites of the two from Merck (Darmstadt, Federal Republic of Germany), tresyl enzymes. chloride was from Fluka (Buchs, Switzerland), Sepharose and DEAE-Sephacel were from Pharmacia (Uppsala, Sweden), and Several coimmobilized multistep enzyme systems have been bis-NAD II (10) N6-[(6-aminohexyl)carbamoylmethyl]-NAD (13) described in the literature (1-6). For example, an immobilized can be obtained from Sigma. bis-NAD I was synthesized ac- system composed of the sequence malate dehydrogenase/ci- cording to the procedure forbis-NAD II (10) but with hydrazine trate synthase showed a consistantly higher overall steady-state instead of adipic acid dihydrazide. bis-NAD III was synthesized rate (6). More recently, a four-enzyme sequence (7) and even by condensing two N6-[(6-aminohexyl)carbamoylmethyl]-NAD the enzymes of a complete metabolic cycle, the urea cycle, have molecules with adipic acid dichloride. The connection with NAD been coimmobilized to supports (8). In the latter case, the im- is through the exocyclic N of adenine. The progress of the syn- mobilized cyclic enzyme system again was more efficient than thesis and the purity of the bis-NAD analogues could be fol- the corresponding soluble system. lowed by HPLAC on a column of silica-bound boronic acid (14). These effects have been partly attributed to the close proximity of the enzymes and partly to the diffusional restrictions H 0 0 H imposed by the Nernst unstirred layer around the enzymes (2, *N-CH2-C-11 NH NH -C-CH2-N* 9). NAD NAD Bifunctional NAD analogues, bis-NAD, have been described as useful reagents for affecting affinity precipitation of enzymes bis-NAD-I (10). In this report we describe the use of such bis-NAD ana- H 0 0 H logues to obtain an immobilized two-enzyme system in which II II the two different active sites are facing one another. The cou- * N-CH2-C-NH-NH- (CH2)6-NH- NH - C - CH2- N* pling of lactate dehydrogenase (L-lactate:NAD' oxidoreduc- NAD NAD tase, EC 1.1.1.27) to immobilized alcohol dehydrogenase (al- bis-NAD-II cohol:NAD' oxidoreductase, EC 1.1.1.1) was carried out with the directing aid of a bifunctional NAD derivative which acted as a template for formation of the two-enzyme complex, before H 0 0 0 0 H the subsequent crosslinking with glutaraldehyde. By such an *N-CH2-C-NH-(CH2)6-NH-C-(CH2)4-C-NH-(CH2)6- NH -C- CH2- N* arrangement, the active sites would be positioned against one NAD NAD another, even after removal of the template, and it could be ex- bis-NAD-III pected that the diffusion of the product of the first enzyme, in this case NADH, to the active site of the second enzyme would Immobilization. Experiment A. Sepharose 4B (2 g of moist be facilitated due to the closer proximity and proper orientation gel) was activated with tresyl chloride as described (15) and about of the active sites, a situation that normally would not occur with 12 mg of alcohol dehydrogenase dissolved in 4 ml of 0.2 M so- soluble enzymes or randomly immobilized species. dium phosphate (pH 7.5) was added. The coupling was allowed to proceed for 2 hr at room temperature, after which the re- The publication costs of this article were defrayed in part by page charge maining active groups on the Sepharose were quenched for 2 hr payment. This article must therefore be hereby marked "advertise- at room temperature with 0.25 M Tris (pH 8.0). After the first ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. immobilization of alcohol dehydrogenase to Sepharose, the gel 1487 Downloaded by guest on September 26, 2021 1488 Biochemistry: Minsson et al. Proc. Nad Acad. Sci. USA 80 (1983) was washed with 0.5 M NaCl/0.2 M sodium phosphate, pH 7.5, NAD that had been used as template during the immobilization and then equilibrated for 10 min at room temperature with 0.2 steps above. The gelwas washed three times with 0.2 M sodium M sodium phosphate, pH 7.5/0.05 M oxalate/0.01 M pyrazole. phosphate, pH 7.5/0.5 M sodium chloride and then with so- Subsequently, about 200 nmol of bis-NAD was added and al- dium phosphate buffercontaining0.1 M isobutyramide, 0.05 M lowed to equilibrate for 10 min in order to form a strong ternary oxamate, and 1 mM NADH. This treatment was carried out be- complex with the pyrazole and the active site of alcohol de- cause it was expected that NADH, togetherwith isobutyramide hydrogenase (Fig. 1, step 1). The gel was subsequently filtered and oxamate, should be capable of forming new ternary com- on a glass filter funnel and the excess bis-NAD was removed by plexes with alcohol dehydrogenase and lactate dehydrogenase, washing with buffer containing pyrazole and oxalate in order to respectively, which should compete with enzyme-bound bis- maintain the ternary complex. The amount of bis-NAD that re- NAD, thereby removing it from the active sites. mained affinity-bound to the alcohol dehydrogenase was cal- In a control experiment, alcohol dehydrogenase and lactate culated by subtracting the bis-NAD removed during washing dehydrogenase were randomly immobilized to tresyl chloride- from that initially added (as determined spectrophotometri- activated Sepharose beads. In this experiment 5.0 mgof alcohol cally). dehydrogenase and 3.0 mg of lactate dehydrogenase were used Lactate dehydrogenase was then added, the amount being per 2.5 g of moist activated gel. the same (in nmol) as that ofthe bis-NAD calculated tobe bound In experiment A the gel was also incubated (Fig. 1, step 5) (Fig. 1, step 2). The lactate dehydrogenase that did not affinity- with bis-NAD (50 nmol/per g of moist gel) in the presence of bind to the bis-NAD pointing out from the active site of alcohol 50 mM oxalate in order to saturate the lactate dehydrogenase dehydrogenase was removed by filtration of the Sepharose beads. active sites with bis-NAD. Excess bis-NAD was removed by Finally, glutaraldehyde, the length of which can vary because washing with 0.1 M sodium phosphate, pH 7.5/50 mM oxalate. of polymerization (16), was added to a final concentration of The amount of bis-NAD that was not affinity-bound upon rein- 0.06%. Coupling (crosslinking) was allowed to proceed for 2.5 cubation was measured (UV absorbance in the filtrate after hr at room temperature (Fig. 1, step 3). All these steps were washing), giving an indirect measurement of the amount that performed in the buffer containing oxalate and pyrazole in order did affinity-bind. to maintain the bis-NAD bound as ternary complex. Experiment B. Alcohol dehydrogenase (2.0 mg) and lipoam- After the glutaraldehyde crosslinking step, the gel was sus- ide dehydrogenase (3.0 mg) were coupled simultaneously to tre- pended in 0.25Tris (pH 8.0) overnight at 40C in order to quench syl chloride-activated Sepharose 4B (2.5 g of moist gel). The the unreacted aldehyde groups of glutaraldehyde. The gel was conditions were the same as for coupling to tresyl chloride-ac- then carefully washed (Fig. 1, step 4) in order to remove the bis- tivated Sepharose 4B in experiment A. Lactate dehydrogenase was then site-to-site immobilized to alcohol dehydrogenase with bis-NAD as template by the same procedure as for site-to-site . ., ADH ADH coupling in experiment A (Fig. 2A). 1 In a control experiment (Fig. 2B), all three enzymes (alcohol, A 2

...- ADH LDH :ADH LDH 3 NAD+ ( NADH,/ NAD+

benzylalcohol ethanof B 5

FIG. 1. Preparation of site-to-site directed alcohol dehydrogenase NADH,' NAD, 1 N4 (ADH)-lactate dehydrogenase (LDH) complex (experiment A). Steps: NAD+ NADH,/ NAD+,' NAD+ 1, affinity binding of bis-NAD to alcohol dehydrogenase immobilized on Sepharose in the presence of pyrazole; 2, affinity binding of lactate dehydrogenase to bis-NAD in the presence of oxalate; 3, crosslinking FIG. 2. Schematic representation of the immobilized three-en- with glutaraldehyde; 4, removal of pyrazole, oxalate, and bis-NAD by zyme system (experiment B). (A) Only alcohol dehydrogenase (ADH) washing; 5, test for site-to-site immobilization by addition of bis-NAD and lipoamide dehydrogenase (LiDH) were coupled directly to tresyl in the presence of oxalate, giving affinity binding to lactate dehydro- chloride-activated Sepharose 4B; lactate dehydrogenase (LDH) was genase and activity in a coupled substrate assay for alcohol dehydro- subsequentlycoupledwiththe site-to-site directingaid of bis-NAD. (B) genase. The oligomeric nature of the enzymes has not been taken into All three enzymes were simultaneously immobilized. The oligomeric account. E2, bis-NAD; 4, pyrazole; *, oxalate. nature of the enzyme has not been taken into account. Downloaded by guest on September 26, 2021 Biochemistry: MAnsson et al. Proc. Natl. Acad. Sci. USA 80 (1983) 1489 lactate, and lipoamide dehydrogenases) were immobilized ran- hydrogenase/lactate dehydrogenase preparations were ob- domly to Sepharose beads; 1.2, 0.6, and 2.0 mg, respectively, tained by using the three different bis-NAD analogues to orient were used per 2.5 g of activated moist gel. the active sites. The object of this first phase in the investigation Enzyme Assays. All assays with immobilized enzymes (25- was to establish whether or not such site-to-site immobilization 100 mg of moist gel) were carried out in a recirculating system could be accomplished and, subsequently, to study the effects using a flow cell (17) mounted in a Hitachi 181 spectrophotome- ofutilizingbis-NAD analogues with different lengths ofthe spacer ter. The total volume in all assays was 15 ml. between the two NAD entities. In experiment A (and its control), the alcohol dehydrogenase Site-to-Site Directed Immobilization. When the immobi- activity was determined in acoupled substrate assay using 5 mM lized alcohol dehydrogenase was incubated with bis-NAD III benzyl alcohol and 5 mM acetaldehyde in order to regenerate (Fig. 1, step 1), 68 nmol of bis-NAD was bound per g of moist the coenzyme in the active site. gel. With the assumption that each of the two active sites bound The required coenzyme NAD was derived from half of the one bis-NAD molecule, this would correspond to 2.7 mg of im- bis-NAD; the other half was affinity-bound to lactate dehydro- mobilized alcohol dehydrogenase. To this 68 nmol of bis-NAD, genase (Fig. 1, step 5). The activity of alcohol dehydrogenase 68 nmol of lactate dehydrogenase was added (Fig. 1, step 2), of was also measured with free NAD (50,uM) in order to obtain the which 30 nmol was bound. The calculations of the amounts of maximal activity. The buffer for activity measurements was 0.2 affinity-bound bis-NAD and lactate dehydrogenase were based M sodium phosphate, pH 7.5/50 mM oxalate in order to keep on UV-absorbance measurements made on the filtrates after the bis-NAD affinity-bound to lactate dehydrogenase during the washing away the excess. The lactate dehydrogenase molecules assay. The activity was recorded as the formation of benzal- added were not affinity-bound to all the bis-NAD bound to aldehyde which has an extinction coefficient of 1,400 cm- M- cohol dehydrogenase, probably due to steric hindrance of the at 279 nm (18). two enzymes. Alcohol dehydrogenase is dimeric, and binding In experiment B, the alcohol dehydrogenase activity was de- of more than one tetrameric lactate dehydrogenase per alcohol termined by using 50 mM ethanol and 1 mM NAD and record- dehydrogenase molecule is probably sterically unfavorable. This ing the NADH production (absorbance at 340 nm). The buffer would imply that the enzyme complex is made up of approxi- was 0.2 M sodium phosphate at pH 7.5. The activities of lactate mately one lactate dehydrogenase molecule and one liver al- dehydrogenase and lipoamide dehydrogenase were determined cohol dehydrogenase molecule connected by one bis-NAD. as follows. Lactate dehydrogenase was measured in 0.2 M so- These steps and the crosslinking were performed in the pres- dium phosphate (pH 7.5) as the decrease in absorbance at 340 ence of oxalate and pyrazole in order to form strong ternary nm with 0.15 mM NADH and 5 mM pyruvate. Lipoamide de- complexes between the enzymes and the bis-NAD and to en- hydrogenase was measured by following the reduction of fer- sure that bis-NAD would remain in the active site. Glutaral- ricyanide (0.75 mM) at 420 nm with 0.2 mM NADH as the sec- dehyde was then used as a crosslinking reagent (Fig. 1, step 3) ond substrate. The extinction coefficient for reduced ferricyanide and, finally, the bis-NAD and the ternary complex-forming agents is 1,040 cm-' M-1. were washed away (Fig. 1, step 4). In the coupled assay for the three-enzyme system in exper- Second Addition of bis-NAD. Reincubation with bis-NAD iment B (and its control), the reduction of ferricyanide (0.75 mM) was performed in the presence of oxalate, leading to strong ter- by lipoamide dehydrogenase was measured with and without nary complex formation with lactate dehydrogenase (Fig. 1, step pyruvate present-i.e., with and without competition between 5). After the excess of bis-NAD was washed away, the active sites lactate dehydrogenase and lipoamide dehydrogenase for NADH oflactate dehydrogenase were assumed to contain one bis-NAD produced by alcohol dehydrogenase (scheme 1). The concen- molecule, one NAD entity affinity-bound in the active site and trations of ethanol and NAD for alcohol dehydrogenase assay the other pointing outward. Because the alcohol dehydrogen- were 50 and 1 mM, respectively; when pyruvate was added, it ase-lactate dehydrogenase complex was formed in the presence was at 5 mM. The amount of NADH oxidized by lactate de- of bis-NAD, the NAD entity that was pointing outward from hydrogenase upon addition of pyruvate was measured indi- lactate dehydrogenase should have been able to reach the active rectly as decrease in lipoamide dehydrogenase activity. site of alcohol dehydrogenase and thus become available as ac- LiDH tive coenzyme. NAD Alcohol Dehydrogenase Activity of the Complexes. The alcohol dehydrogenase activities of the crosslinked enzyme com- Ferricyanide Ferrocyanide plex (alcohol dehydrogenase-lactate dehydrogenase) obtained under different conditions are given in Table 1. There was some NAD ADH , NADH activity even prior to incubation with bis-NAD. This probably was due to the presence of some bis-NAD from the preceding Ethanol Acetaldehyde crosslinking treatment that had not been washed away. It may be that the crosslinking physically entrapped these bis-NAD LDH NAD molecules between the two active sites. As much as 45% of the alcohol dehydrogenase activity ob- Pyruvate Lactate tained in the presence of excess NAD (50 ,tM; Km for soluble enzyme, 17 tkM) could be obtained by incubation with bis-NAD ADH, alcohol dehydrogenase; LDH, lactate dehydrogenase; III, which is recycled in the active site of alcohol dehydrogen- LiDH, lipoamide dehydrogenase. ase. The nominal concentration of NAD available for alcohol Scheme 1 dehydrogenase was only 0.05 ,uM (concentration of bis-NAD). available NAD entity of the other bis-NAD an- The sterically-II RESULTS alogues also allowed alcohol dehydrogenase activity but to a lesser extent. Crosslinking performed in the presence of bis-NAD II Confirmation of site-to-site immobilization (Table 1) and of bis-NAD I (data not shown here) gave the same results. However, less activity was obtained after readdition of In experiment A, crosslinked site-to-site directed alcohol de- the bis-NAD analogues. The general conclusion that can be drawn Downloaded by guest on September 26, 2021 1490 Biochemistry: MAnsson et al. Proc. Natl. Acad. Sci. USA 80 (1983) Table 1. Activity of immobilized alcohol dehydrogenase-lactate dehydrogenase complex Alcohol dehydrogenase activity, ,umol/min per 100 mg moist gel* Prior to After reincubation Conditions reincubation bis-NAD I bis-NAD II bis-NAD III Crosslinking in No soluble NAD added 0.134 0.321 0.70 1.02 presence of NAD (50 pM) added 1.84 2.05 1.96 2.27 bis-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 0.411 presence of NAD (50 IM) added 1.69 1.38 1.66 1.66 bis-NAD II Activity ratio 11.2 17.8 32.4 24.8 Randomly No soluble NAD added 0 0.069 0.058 0.054 coupled 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 to lactate dehydrogenase or soluble NAD as coenzyme. Total assay volume was 15 ml with 5 mM benzyl alcohol and 5 mM acetaldehyde 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- bilization step, alcohol dehydrogenase and lipoamide dehydro- drogenase results with use of those bis-NAD analogues used in genase were coimmobilized randomly to tresyl chloride-acti- the actual crosslinking of the two enzymes. vated Sepharose 4B; then lactate dehydrogenase was coupled to Random Coimmobilization. In a control to experiment A, al- alcohol dehydrogenase with the site-to-site directing aid of bis- cohol dehydrogenase and lactate dehydrogenase were randomly NAD III. coimmobilized on tresyl chloride-activated Sepharose 4B. This When ethanol and NAD were added to such a system, acetal- preparation was then incubated with the different bis-NAD an- dehyde and NADH were formed by alcohol dehydrogenase. The alogues in the same way as the gels with the site-to-site directed NADH that was produced could be reoxidized to NAD either immobilized enzymes. Much less activity (4-5%) was observed by lipoamide dehydrogenase or by lactate dehydrogenase (Fig. upon readdition of bis-NAD when the enzymes were randomly 2). With no pyruvate present (i.e., no lactate dehydrogenase immobilized (Table 1), most likely because the free end of the activity), the NADH produced by alcohol dehydrogenase could bis-NAD affinity-bound to lactate dehydrogenase did not reach only be oxidized by lipoamide dehydrogenase. The activity was to alcohol dehydrogenase since the enzyme active sites were too measured as the decrease in absorbance at 420 nm due to fer- far apart. ricyanide reduction. When pyruvate was added, lactate dehy- The amount of bis-NAD that did affinity-bind to lactate de- drogenase started to compete with lipoamide dehydrogenase for hydrogenase upon readdition of bis-NAD to the two-enzyme the produced NADH. The amount of NADH oxidized by lac- complex of alcohol dehydrogenase and lactate dehydrogenase tate dehydrogenase and lipoamide dehydrogenase, respec- was roughly the same for all three bis-NAD analogues, both for tively, would be expected to be determined by the relative total site-to-site directed coimmobilization and for random coim- numbers of enzyme units of the two enzymes. This was also found mobilization. This shows that the lower activity observed upon to be the case for the randomly coupled three-enzyme system bis-NAD readdition in the control experiment was not the result (Fig. 2B) in which the amount of NADH oxidized by lactate de- of a lower bis-NAD concentration. hydrogenase was roughly the expected one, 0.5% found and 4.5% expected (Table 2). With the same assumption, 19% of formed Immobilized three-enzyme system NADH would be expected to be oxidized by lactate dehydrogenase in the system of the same three enzymes but with lactate In order to study the effects of juxtaposition of the active sites dehydrogenase juxtaposed to alcohol dehydrogenase. How- of alcohol dehydrogenase and lactate dehydrogenase, a third ever, as much as 50% of the formed NADH was oxidized by lac- enzyme, lipoamide dehydrogenase, was incorporated into the tate dehydrogenase which indicates that the NADH was. pref- system as a "reporter enzyme" (experiment B). This was chosen erentially channeled from alcohol dehydrogenase directly to because it is able to compete with lactate dehydrogenase for lactate dehydrogenase. The net effect was that much less NADH NADH formed by alcohol dehydrogenase. In the first immo- became 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 LDH ADH LiDH LDH Theoretical Found Theoretical Found Site-to-site coupling 0.018 1.1 0.26 81 50 19 50 Random 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) of the immobilized three-enzyme systems were first measured under their V.. conditions. From these activities the theoretical values 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 decrease in LiDH activity upon addition of pyruvate. Downloaded by guest on September 26, 2021 Biochemistry: MAnsson et aL Proc. Nati Acad. Sci. USA 80 (1983) 1491 DISCUSSION nase to glucose-6-phosphate dehydrogenase has been used to ExperimentA. From the-data in Table 1, it becomes apparent obtain a more efficient assay system (21). A site-to-site arrange- that one end of the bis-NAD analogues tested can interact with ment possibly could further improve the assay. Site-to-site im- alcohol dehydrogenase while the other half of the molecule is mobilized enzymes might also be useful for improving the en- affinity-bound (in a ternary complex) to lactate dehydrogenase. zymic regeneration of coenzymes in a system like the one This implies that the overall geometry of the two enzymes rel- described (22) in which the coenzyme NAD was covalently cou- ative to one another, obtained upon crosslinking, is retained also pled to alcohol dehydrogenase and regenerated with soluble after the bis-NAD template has been washed away. The ge- lactate dehydrogenase. ometry is even retained to the extent that the bis-NAD analogue that was used in the cross-linking yields the highest activity of We-thank Professor Per-Olof Larsson for valuable advice concerning alcohol dehydrogenase, thus indicating the best fit. The results the synthesis of the three bis-NAD analogues and the Swedish Natural from a blank experiment (Table 1) in which the two enzymes had Science Research Council (Grant 2616-107) and the National Board for been randomly coupled to Sepharose show that the bis-NAD Technical Development (Grant 80-3595) for their generous financial analogues added in this case do not interact to any large extent support. with alcohol dehydrogenase under the conditions used. This is 1. Mosbach, K. & Mattiasson, B. (1970) Acta Chern. Scand. 24, 2093- most likely due to the fact that the two enzymes are too far apart 2100. or are not properly oriented toward one another due to the ab- 2. Mattiasson, B. & Mosbach, K. (1971) Biochim. Biophys. Acta 235, sence of the bis-NAD analogue during coupling. 253-257. Experiment B. The objective of this study was to investigate 3. Mosbach, K. & Mattiasson, B. (1978) in Current Topics in Cel- whether by such orientation the "product" i.e., NADH-of lular Regulation, eds. Horecker, B. L. & Stadtman, E. R. (Aca- alcohol dehydrogenase would preferentially be channeled to demic, New York), Vol. 14, pp. 197-241. 4. Lecoq, D., Hervagault, J. F., Broun, G., Joly, G., Kernevez, J. lactate dehydrogenase or to a competing enzyme, such as lipo- P. & Thomas, D. (1975)J. Biol Chem. 250, 5496-5500. amide dehydrogenase, bound in vicinity ofthe enzyme complex 5. Jablonski, E. & DeLuca, M. (1978) Methods Enzymol. 57, 202- on the same Sepharose beads. Interpretation of the results ob- 214. tained was simplified because the Km values for NADH of lac- 6. Srere, P., Mattiasson, B. & Mosbach, K. (1973) Proc. Nati Acad. tate dehydrogenase and lipoamide dehydrogenase are about the Sci. USA 70, 2534-2538. same, 23 (19) and 25 (unpublished data), respectively. 7. Okamoto, H., Tipayang, P. & Inada, Y. (1980) Biochim. Biophys. AM ,AM Acta 611, 35-39. The sum of the lactate dehydrogenase and lipoamide dehydro- 8. Siegbahn, N. & Mosbach, K. (1982) FEBS Lett. 137, 6-10. genase activities present was much greater than the alcohol de- 9. Goldman, R. & Katchalski, E. (1971)J. Theor. Biol 32, 243-257. hydrogenase activity, which led to the rapid oxidation, by either 10. Larsson, P.-O. & Mosbach, K. (1979) FEBS Lett. 98, 333-338. of the two enzymes, of the NADH produced by alcohol de- 11. Ovadi, J., Salerno, C., Keleti, T. & Fasella, P. (1978) Eur. J. hydrogenase. Thus, neither lactate dehydrogenase nor lipoam- Biochem. 90, 499-503. ide dehydrogenase was operating under conditions. The 12. Ovadi, J. & Keleti, T. (1978) Eur. J. Biochem. 85, 157-161. Vm. 13. Lindberg, M., Larsson, P.-O. & Mosbach, K. (1973) Eur. J. results obtained definitelyprove that-preferential channeling of Biochem. 40, 187-193. NADH to lactate dehydrogenase does occur (50% oxidation by 14. Glad, M., Ohlson, S., Hansson, L., MAnsson, M.-O. & Mosbach, LDH instead of the theoretical value of 19%). K. (1980) J. Chromatogr. 200, 254-260. Conclusions. With the aid of bis-NAD analogues, or possibly 15. Nilsson, K. & Mosbach, K. (1981) Biochem. Biophys. Res. Com- also with other bis compounds such as bis inhibitors, crosslinked mun. 102, 449-457. enzyme complexes can be obtained in an oriented way. At the 16. -Peters, K. & Richards, F. (1977) Annu. Rev. Biochem. 46, 523- same time, the active sites are protected during coupling with 551. -17. Mattiasson, B. & Mosbach, K. (1976) Methods Enzymol 44, 335- the aid of such orienting molecules, and the enzymes are brought 353. into close and directed.(active sites juxtaposed) proximity. Such 18. Fuller, C. W, Rubin, J. R. & Bright, H. J. (1980) Eur.J. Biochem. preparations may serve as valuable model systems for consec- 103, 421-430. utively operating enzyme systems in which preferred channel- 19. Winer, A. & Schwert, G. W. (1958) J. Biol Chem. 231, 1065-1083. ing of the intermediates occurs, or for bifunctional enzymes like 20. Kirschner, K. & Wiskocil, R. L. (1972) in Protein-Protein Inter- tryptophan synthase (20) with which the product of one subunit actions, eds. Jaenicke, R. & Helmreich, E. (Springer, Berlin), pp. 245-268. reaction, indole, is channeled to the second subunit for reaction 21. Litman, D., Hanlon, T. & Ullman, E. (1980) AnaL Biochem. 106, with. serine leading to tryptophan. In a specific homogeneous 223-229. enzyme immunoassay technique (enzyme channeling immu- 22. Minsson, M.-O., Larsson, P.-O. & Mosbach, K. (1979) FEBS Lett. noassay), the channeling of glucose 6-phosphate from hexoki- 98, 309-313. Downloaded by guest on September 26, 2021