Site-To-Site Directed Immobilization of Enzymes with Bis-NAD Analogues
Total Page：16
File Type：pdf, Size：1020Kb
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 prox- imity 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+ n4 benzylalcohol ethanof B 5 FIG.