Role of lysine residues in the nucleotides binding to bovine liver high-Km Aldehyde reductase

Bid. Vol. 27. NO. 5, pp. 457-467. 1995 Convrieht I( 1995 Elsevier Science Lid --r, ” Printed in Grea; Britain. All rights reserved 1357-2725/95 59.50+0.00

In/. J. Biochem. Cd

Pergamon

1357-2725(%)00020-8

Role of Lysine Residues in the Nuckotides Bovine Liver High-Km AIdehyde Redwtase TOMOYUKI

TERADA

Laboratory of Biochemistry, Faculty of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita, Osaka 56.5, Japan The inactivation of bovine liver high-K,,, akiehyde reductase (ALR) by heat (47’C), 0.3 mM 2,4,6-triuitrobenzeue sulfonate (TNBS) and 0.03 mM pyridoxal Y-phosphate (PAL-P) followed pseudo-first-order kinetic as a function of incubation-time and concentration of TNBS. Nucleotides which have a Z’qhosphate group, especially /I-NADPH and fi-NADP+, showed effective protection on ALR-inactivation. However, typical substrates for ALR such as D,L-glycerahkhyde, wrythrose, D-glucuronate and p-carboxyhenzakkhyde could not protect the enzyme from inactivation. Completely inactivated enzyme was estimated to have 2.07 TNBS-mod&d lysine residues/mol enzyme from the determination of free amino group using fluorescamine (Ex = 390 nm, Em = 475 nm). Enzyme protected by fl-NADP+ (96.5% remaiuiug activity) did not lose a significant number of lysine re&htes. Kd-values for /?-N ADPH aud /I-NADP+ were estimated to be 0.48 pM and 4.7 CM, respectively and TNBS-treated enzyme lost its ability to bind to these nucleotides. Keywords: Aldehyde reductase Lysine residue Nucleotide binding Bovine liver Int. J. Biochem. Cell Biol. (1995) 27, 457-467

this aldehyde reductase is as follows; a specificity for /I?-NADPH and strong activities for D-glucuronate, short chain aldoses (D,L-glyceraldehyde, D-erythrose and D-erythrose 4-phosphate), aromatic aldehydes @-carboxybenzaldehyde, p-nitrobenzaldehyde, pyridine 3-aldehyde and pyridine 4-aldehyde), drugs (daunorubicin and acetohexamide) and tramdihydrodiols (trans - 1,2-dihydro- 1,2-benzenedihydrodiol and trans-l,2-naphthalenedihydrodiol) with high &,-values (Wermuth et al., 1977; Felsted and Bachur, 1980; Morepeth and Dickinson, 1981; Vogel et al., 1982; Terada et al., 1985; Mizoguchi et al., 1992). In the course of our preceding studies, we have reported the presence of three dihydrodiol dehydrogenases (DDs) in bovine liver cytosol (DDl, DD2 and DD3) and they were identified as 3u-hydroxysteroid dehydrogenase (EC 1.1.1.50), high-K,,, aldehyde reductase and dihydrodiol specific enzyme (Nanjo et al., 1992; Mizoguchi et al., 1992; Nanjo et al., 1993), respectively. Among these DDs, we also suggested both DDl and DD3 had an essential cysteinyl residue(s) for activity in their molecule

INTRODUCTION

Aldo-keto reductases are classified into three major classes, aldehyde reductase (EC 1.1.1.2), aldose reductase (EC 1.1.1.21) and carbonyl reductase as proposed by Turner and Flynn in 1982 in the aspects of substrate specificity, inhibitor sensitivity and cofactor specificity (Turner and Flynn, 1982). Many enzymes from various animals and tissues have been suggested to belong to the category of aldehyde reductase, i.e. high-K, aldehyde reductases (EC 1.1.1.2), glycerol dehydrogenases (EC 1,1.1.72), D-glucuronate reductases (EC 1.1.1.19) and dihydrodiol dehydrogenases (EC 1.3.1.20) (Wermuth et al., 1977; Felsted and Bachur, 1980; Morepeth and Dickinson, 1981; Vogel et al., 1982). The most characteristic properties of Abbreuiarions:ALR, aldehyde reductase; DD, dihydrodiol dehydrogenase; TNBS, trinitrobenzene sulfonate, PAL-P, pyridoxal S-phosphate; DTNB, W-dithiobis(2-nitroknzoic acid); b-NADP+, acetyl-fl-NAD(P)+; ADP-R-phosphate, ADP-ribose phosphate; j-NMN, nicotineamide mononucleotide; pCBA, p-carboxybenzaldehyde. Received 5 August 1994; accepted 3 February 1995. 451

458

Tomoyuki

and also proposed the modulation of their activities through thiol/disulfide exchange reaction as similar to rat liver DD (Terada et al., 1993). On the other hand, DD2 was insensitive to SH-reagents such as 0.1 mM 5,5’-dithiobis(2-nitrobenzoic acid), 0. I mM N-ethylmaleimide, 5 mM GSSG and I mM cystine (Mizoguchi et al., 1992). Flynn et al. suggested the presence of lysine residue(s) in the active site of pig kidney high-K, aldehyde reductase as similar to the case of lactate dehydrogenase (Davidson and Flynn, 1979; Flynn et al., 1981). Higuchi et al. also investigated the possibility of the presence of lysine residue in the vicinity of cofactor binding site of carbonyl reductase (Higuchi et al., 1994) as similar to the cases of bovine liver glutamate dehydrogenase (Anderson et al., 1966), rabbit muscle glyceraldehyde 3-phosphate dehydrogenase (Ranch et al., 1969) and rat liver cytochrome P-450 reductase (Inano and Tamaoki, 1986). In the present study, I demonstrated that the 2 lysine residues in bovine high-K,,, aldehyde reductase play an important role in the binding to pyridine nucleotides using chemical modification techniques with 2,4,6-trinitrobenzene sulfonate (TNBS) and pyridoxal 5’-phosphate (PAL-P), and thermal treatment at 47°C. MATERIALS

AND

METHODS

Materials

/3-NADPH, /?-NADH, /?-NADP+ and /3-NAD+ were purchased from Oriental Yeast Co., Tokyo. The other nucleotides, substrates and a-hippuryl-L-lysine were obtained from Sigma Chemical Company, St Louis, MO. 2,4,6-Trinitrobenzene sulfonate (TNBS), pyridoxal 5’-phosphate and L-leucine were the products of Wako Pure Chemical Industries, Osaka. Fluorescamine was supplied from Nacalai Tesque, Kyoto. The other reagents were the highest grade commercially available. The enzyme was prepared according to the method of our previous paper using ammonium sulfate fractionation and some column chromatowith DEAE-cellulose, graphic techniques Ultrogel AcA 44, Blue-cellulofine, 2’,5’-ADPSepharose and hydroxyapatite (Mizoguchi et al., 1992). Methods Assay of enzymatic activity. The enzymatic activity of ALR was performed essentially according to the method of previously described

Terada

(Terada et al., 1985) under the following conditions; 100 mM phosphate buffer, pH 7.0, 5.0mM p-glucuronate and 0.065 mM fiNADPH at 25°C monitoring the decrease of absorbance at 340 nm. Heat-treatment of ALR. ALR (lOO~gg/ml) in 100 mM phosphate buffer, pH 7.0, was inactivated by the treatment at 47°C with/ without nucleotides or substrates. The samples were taken out for the determination of the remaining activity at appropriate intervals. Inactivation of ALR with 2,4,tGtrinitrobenzene sulfonate (TNBS) and pyridoxal 5’-phosphate (PAL-P). Chemical modifications of ALR with

TNBS and PAL-P were carried out at 25°C in the following reaction mixtures; 60 mM phosphate buffer, pH 7.0,0.3 mM TNBS or 0.03 mM PAL-P and 45 pgg/ml of ALR with/without nucleotides or substrates. At appropriate intervals, the samples were taken out for the determination of remaining activity. To avoid photosensitizing inactivation of ALR with PAL-P, the modification was performed in the tube with black plastic cover. Determination

of modtfied

lysine residues.

Measurement of the modified lysine residues in ALR molecule was performed according to the method of Bohlen (Btihlen et al., 1973) as described below. The determinations of the emission at 475 nm (excitation at 390 nm) were carried out with Hitachi model F-3000 fluorescence spectrophotometer. The reaction mixture contained 100 mM sodium borate buffer, pH 9.0, 12 PM of fluorescamine (dissolved in acetone) and appropriate amount of sample. L-Leucine and cl-hippuryl-L-lysine were used as standards. Titration of p-NADPH or j?-NADP+ binding with ALR. The determination of /?-NADPH or P-NADP+ binding with ALR (12.8pM in 100 mM phosphate buffer, pH 7.0) was carried out monitoring the increase of the absorbance at 380 or 360 nm with the addition of various concentrations of pyridine nucleotides into the enzyme solution, respectively. RESULTS

Inactivation of ALR by heat-treatment of various compounds

and eflect

Inactivation of ALR by heat-treatment was advanced as a function of incubation-time (data not shown). The effects of nucleotides and substrates on this inactivation of ALR were tested with/without nucleotides or substrates listed

Lysine residues in bovine liver ALR

159

Table I. Protective effect of various nucleotides on the thermal-inactivation of bovine liver high-K,,, aldehyde reductase (ALR). (Details described in the section of “Methods”). After heat-treatment at 47°C for IO min, the enzyme (100 wg/ml) was subjected to the determination of remaining activity. The values in this table represent the average of 5 experiments Remaining Concentration activity f SD (%) Compound (PM) 41.6 + 4.78 No addition 98.0 + 3.33 100 /I-NADPH 92.4 i 2.98 IO 87.5 + 6.22 100 fl-NADP+ 70.7 + 3.86 IO 100 43.6 rf 3.38 fi-NADH 100 36.6 + 6.12 fi-NAD+ 100 98.4 + I .46 Acetyl-fi-NADP+ I00 43.2 + 3.83 Acetyl-P-NAD+ 100 86.2 k 4.95 ADP-R-P 100 87.0 + 4.27 2’,5’-ADP 100 63.3 f 2.01 2’-AMP 100 43.5 &- 5.23 5’-AMP 100 45.5 &-4.66 5’-ADP 100 50.7 k 3.98 5’-ATP 47.6 + I .96 3’,5’-CAMP 100 100 45.0 k 2.94 3’-AMP 41.6 + 3.03 /?-NMN 100 32.1 + 4.21 o,L-Glyceraldehyde IOmM 39.9 + 4.01 o-Erythrose IOmM 45.8 k 2.78 o-Glucuronate IOmM p-Carboxybenzaldehyde ImM 44.6 + 2.99 2-Mercaptoethanol 50 mM 38.7 + 2.37 Dithiothreitol IO mM 39.5 + 2.11 ATP, adenosine triphosphate; ADP, adenosine diphosphate; AMP, adenosine monophosphate; ADP-R-P, ADP-ribose phosphate; NMN, nicotineamide mononucleotide.

in the Table 1 at indicated concentrations. The results in the Table 1 show J?-NADPH can protect ALR activity completely at lower concentration (10 PM) than those of the other nucleotides (100 p M). b-NADP+ (10 PM) shows less effectiveness on the protection of ALR than j?-NADPH, but, still more significant than the other nucleotides. Additionally, ALR was also protected by ADP-R-P, 2’,5’-ADP or 2’-AMP efficiently at 1OOpM. However, the other nucleotides such as /.I-NADH, B-NAD+, 5’-AMP and nicotineamide mononucleotide (j?-NMN) which are components of j?-NADP(H) did not show any protection on ALR-inactivation. Furthermore, nucleotides which have no 2’-phosphate group including 5’-ADP, 5’-ATP, 3’,5’-CAMP and 3’-AMP were insignificant on the protection of ALR. On the other hand, substrates (D-erythrose, D,t-glyceraldehyde, o-glucuronate and p-carboxybenzaldehyde) and thiol compounds (dithiothreitol and 2-mercaptoethanol) did not have any ability to protect ALR completely at high concentrations (data not shown). inactivation

of ALR by TNBS-treatment

The inactivation of ALR with TNBS was extended as functions of the incubation-time

and concentrations of TNBS as shown in Fig. 1. The result (n = 1.08) of kinetic analysis according to the method of Levy (Levy et al., 1963) (log k, = log k, + n log [TNBS]) suggested that this inactivation followed the psuedo-first order kinetic manner (k, and k, indicate the first-order reaction rate constant and the secondary-order reaction rate constant, respectively). To clarify that the inactivation of ALR was caused by the modification of lysine residues and not cysteine residues, 0.5 mM 5,5’-dithiobis(2nitrobenzoic acid) (DTNB)-treatment of ALR was carried out. DTNB-treated ALR kept its full activity (97.2% remaining activity) as compared with no-treated ALR, suggesting no involvement of cysteine in ALR-activity. Inactivation of ALR with PAL-P treatment PAL-P is a well-known modification reagent of lysine residue in the protein as well as TNBS. This compound is supposed to be fixed with a s-amino group of lysine through schiff base formation in the presence of sodium borohydride and also plays a photosensitizer similar to various dyes (rose bengal and methylene blue). To avoid this photosensitization, ALR was treated with PAL-P in the tube with black plastic cover. ALR was potently inactivated by

460

Tomoyuki Terada

25

t

--

0.0 0.1 0.2 0.3 0.4 0.5

Cont. (mM) 0

20

40

60

Incubation

time (mm)

80

100

Fig. 1. Inactivation of ALR as functions of incubation-time and concentration of TNBS. The enzyme (40 pg/ml) in 100 mM phosphate buffer, pH 7.0, was incubated with various concentrations of TNBS at 25°C; l . 0 mM; 0.0.05 mM; A, 0.1 mM; A, 0.2 mM; n , 0.3 mM; iJ,O.4 mM and +, 0.5 mM. Aliquots samples were taken out for determination of their remaining activity following the incubation time. Secondary plots (slope vs concentration) replotted in the inserted figure.

the treatment with PAL-P at a lower concentration than that of TNBS without sodium borohydride as shown in Table 2. Eflect of pH on the TNBS-inactivation

of ALR

It is well-known that chemical modification of residues in the enzymes is advanced depending on the charge of residues. Figure 2 showed that the pseudo-first-order-rate constants obtained from TNBS-inactivation of ALR under various pHs were clearly followed pH, namely, the enzyme was inactivated rapidly under high pHs. Eflect of various nucleotides and compounds on the protection of ALR from TNBS- and PAL-P mod@cations As shown in Table 2, the effects of various compounds including nucleotides, substrates and thiols as listed in the Table on the modifications by TNBS and PAL-P were tested. Similar to the results of heat-treatment, both P-NADPH and /?-NADP+ were the potent protectors for both modifications. Either 2’,5’-ADP or 2’-AMP was also able to protect ALR very effectively. ADP-R-P also showed an effective protection on the activity of

ALR. Furthermore, acetyl-p-NADP+, as an analogue of /3-NADP(H), was also an efficient protector for ALR inactivations. However, the other nucleotides as listed in the Table had no ability to protect ALR at all as in the case of heat-treatment. In addition, substrates, D-erythrose, D,L-glyceraldehyde, D-glucuronate and p-carboxybenzaldehyde, were not protective to ALR-inactivation (data not shown). Effect of concentrations on these compounds on ALR-protection from TNBS-treatment was further investigated. As shown in Fig. 3(A), p-NADPH was the most potent protector for ALR-inactivation, then B-NADP+. Both /I-NADH and fl-NAD+ were less effective even at lOOO- to lO,OOO-fold higher concentrations than those of B-NADPH and /?-NADP+. On the other hand, substrates showed an insignificant effect on the protection of ALR activity [Figure 3(B)]. Determination of lysine residues of TNBS modljied ALR The number of modified lysine residues in ALR were determined by fluorescamine method (Biihlen et al., 1973). As shown in Fig. 4, the

461

Lysine residues in bovine liver ALR

number of lysine residues were significantly decreased coincident with the inactivation of ALR, suggesting that the modification of 2 mol lysine residues/mol enzyme leads ALR to be completely inactivated in the absence of /I-NADP+ . A single phase kinetic manner in the modification of lysines vs remaining activity of ALR suggested that the modification of these lysine residues with TNBS were advanced with same rate constants. On the other hand, no significant loss of lysine residue of ALR (0.17 mol lysine/mol enzyme) with TNBS-treatment was observed in the presence of 1 mM /I-NADP+ as shown in Table 3. Additionally, loss of 2 lysine residues were also observed in the TNBS-treated ALR in the presence of 1 mM b-NAD+ or 10mM D-erythrose (Table 3). Efect of heat-treatment and chemical modiJication on /3-NADPH and fl-NADP+ binding with ALR Figure 5 shows the results of the difference absorption spectrum of ALR with pyridine Table 2. Effect of nucleotides on the inactivation of bovine liver high-K,,, aldehyde reductase (ALR) by 2,4,6-trinitrobenzene sulfonate (TNBS) and pyridoxal 5’-phosphate (PAL-P). The enzyme (45pg/ml) was incubated with 0.3 mM TNBS or 0.03 mM PAL-P in the presence or absence of nucleotides (1 mM), thiols (50mM 2-mercaptoethanol and 10 mM dithiothreitol) and substrates (10 mM o-erythrose, o,L-glyceraldehyde and o-glucuronate, and 1 mM p-carboxybenzaldehyde) at 25°C for 80 min. (For details see text). The values in this table indicate the averages of 4 experiments Remaining activity + SD (X) Nucleotide TNBS PAL-P None 39.4 f 4.58 48.8 + 3.94 /?-NADPH 96.1+ 2.63 95.6 + 2.56 /I-NADP+ 91.2 + 3.89 91.4* 4.10 &NADH 63.3 + 4.41 78.7 k 3.68 $NAD+ 53.1 f 1.67 64.7 + 2.11 Acetvl-B-NADP+ 88.7 k 4.25 88.4 + 3.33 Ace&l-b-NAD+ 47.0 _+ 3.33 50.0 f 4.67 ADP-R-P 93.3 * 5.03 93.8 & 2.10 2’,5’-ADP 95.2 + 2.61 96.6 k 2.59 2’-AMP 59.4 k 3.42 68.1 & 3.80 5’-AMP 48.7 _+ 3.38 65.2 _+2.59 5’-ADP 73.8 + 1.36 74.0 + 4.39 5’-ATP 71.1 + 2.95 86.0 * 1.34 /?-NMN 47.3 k 1.29 58.3 + 4.44 Adenosine 44.7 k 2.88 49.6 + 5.01 o,L-Glyceraldehyde 37.2 + 2.27 42.5 f 1.66 u-Erythrose 42.5 + 3.66 48.5 k 3.07 o-Glucuronate 50.1 * 2.21 55.6 + 3.83 p-Carboxybenzaldehyde 52.9 _+ 3.98 57.2 _+ 2.94 2-Mercaptoethanol 38.0 + 4.47 46.8 + 2.67 Dithiothreitol 39.1 + 2.67 44.1 * 3.34 TNBS, Trinitrobenzene sulfonate; PAL-P, pyridoxal 5’-phosphate; ATP, adenosine triphosphate; ADP, adenosine diphosphate; AMP, adenosine monophosphate; ADP-R-P, ADP-ribose phosphate; NMN, nicotineamide mononucleotide.

nucleotides, /I-NADP(H). When /I-NADPH was used, the spectrum showed the decrease of absorbance at around 340 nm and the increase of absorbance at around 380 nm with the addition of /I-NADPH (data not shown). On the other hand, in the case of /?-NADP+, only the increase of absorbance at around 360 nm was observed with the addition of P-NADP+ (data not shown). Titration analysis of /I-NADPH or fi-NADP+ binding with ALR molecule, monitoring the increase at 380 or 360 nm, showed that the apparent Kd-value for /I-NADPH or /I-NADP+ was calculated to be 0.48 or 4.7 PM, respectively. However, pyridine nucleotides which have no 2’-phosphate, /I-NADH or fl-NAD+, showed a very similar absorption spectrum but smaller than that of /I-NADPH or /I-NADP+, respectively (data not shown) due to their lower affinity for ALR (300-fold higher &,-value compared with that for fl-NADPH). The inactivated ALR lost its binding ability to these pyridine nucleotides such as /I-NADPH or fl-NADP+ (data not shown). Following our expectation, fl-NMN can not bind to ALR by itself from the result of the absorption spectrum. DISCUSSION

It has been suggested that lysine and arginine residues have an important role in the catalytic activity of aldehyde reductases (Morjana et a/.,1989; Bohren et al., 1991) using chemical modification reagents and site-directed mutagenesis techniques similar to other enzymes including the other aldo-keto reductases (Morjana et al., 1989; Bohren et al., 1991; Penning et al., 1991; Yokoyama et al., 1992; Higuchi et al., 1994; Kubiseski et al., 1994), fructose 1,6-diphosphatase (Colombo and Marcus, 1974), glucose 6-phosphate dehydrogenase (Milhausen and Levy, 1975; Jeffery et al., 1985) and pyruvate phosphate dikinase (Phillips et al., 1983). The results of the studies on aldose reductase suggested that Lys262 has been investigated to participate in coenzyme binding (Morjana et a/.,1989; Bohren et al., 1991) using chemical modification and site-directed mutagenesis techniques. It has been reported that -Ile-Pro-Lys 262-Ser-motif is conserved in the active site of all aldo-keto reductase superfamilies (Bohren et al., 1989, 1991; Penning et al., 1991; Yokoyama et al., 1992) and it locates near C-terminal region of aldose reductase from the result of crystallographic

462

Tomoyuki Terada

20 40 60 Incubation Time (min)

6.0

6.5

7.0

7.5

8.0

8.5

9.0

9.5

PH Fig. 2. Effect of pHs on the pseudo-first-order rate constants of the inactivation of ALR with TNBS. The enzyme (40 pg/ml) was incubated at 25°C with 0.3 mM TNBS under various pHs using followed buffers; pH 6.0-8.0, 100mM NaH,PO,/Na,HPO, buffer (0); pH 8.0-9.0, IOOmM Na2B,0,/KH,P0, buffer (A); pH 9.0-10.4, 100mM Na,CO,/NaHCO, buffer (m). The obtained pseudo-first-order rate constants of the inactivations of ALR at pHs were replotted in the figure. Inserted figure; 0, pH 9.6; 0, pH 9.0; 0, pH, 8.5; +, pH 8.0; V, pH 7.5; V, pH 7.0; f, pH 6.5.

study (Rondeau et al., 1992), meaning this motif may be an important region for activity. The same motif (-Ile-Pro-Lys26’-Ser-) was also conserved in the deduced amino acid sequence of human ALR cDNA as reported (Bohren et al., 1989, 1991). On the basis of these reports, the study of the role of lysine residue(s) in bovine liver cytosolic aldehyde reductase has been investigated in the present study using chemical modification techniques with TNBS and PAL-P, and analysis of absorption spectrum. As shown in Fig. 1, the results of the inactivation of ALR by TNBS suggests that TNBS reacts on two lysine residues in the enzyme molecule with same rate. Additionally, the results of the slope (remaining activity vs incubation-time) which were calculated from the inactivation by TNBS under various pHs replotted in Fig. 2 showed that the inactivation of ALR was highly advanced, depending on pHs with a clear sigmoid curve, suggesting the involvement of a single species of amino acid residue in the inactivation of ALR. An apparent inflection point of this curve at pH 8.8 suggests the involvement of lysine residue (PI-value, 8-9) in the inactivation. The results in Table 2 showed both TNBS and PAL-P, well-known

as modification reagents of c-amino group of lysine, participate in the inactivation of ALR with a similar manner. Furthermore, the study on the protection of ALR from the inactivation under various concentrations of compounds as listed in the Fig. 3 showed that reduced forms of nucleotides have greater affinities for the enzyme than the oxidized form, regardless of 2’-phosphate [Kd-value for @-NADPH (0.48 PM) is much higher than that of p-NADPH (4.7 PM)]. Though pCBA seems to have a significant protection in Fig. 4, the concentration (5 mM) is 600- to 700-fold higher compared with its &,-value (8 PM), supposing pCBA to be a less effective protector for the inactivation of ALR than NADP(H). Considering these results of the effects of nucleotides and substrates on the inactivation of ALR, it is suggested that the lysine residue(s) locates nearby/in the nucleotide binding site, not the substrate binding site as similar to the previous papers (Davidson and Flynn, 1979; Flynn et al., 1981; Davidson, 1987; Bohren et al., 1991). Additionally, the following result suggests that lysine residue(s) has a possible role in the tight binding of 2’-phosphate of nucleotides with the enzyme molecule; the nucleotides which have 2’-phosphate such as /?-NADPH, P-NADP+,

Lysine

residues

in bovine

acetyl-P-NADP+ , ADP-P-R and 2’S’-ADP showed a highly protective effect on the inactivation, but I-NADH, fl-NAD+, 5’-ADP and p-NMN also showed no effectiveness at all. The speculations of important involvement of 2’-adenylate moiety and weak involvement of 5’-phosphate in the binding with ALR were also strongly supported by the results that 2’-AMP is a less effective protector on the inactivations of ALR than 2’,5’-ADP. The modified lysine residue(s) was determined by fluorescamine method (BGhlen et al., 1973)

liver

463

ALR

monitoring the decrease of total lysine residues using CI-hippuryl-L-lysine and L-leucine as standards of E- and r-amino groups, respectively. Figure 5 showed that the enzyme was completely inactivated when it lost 2 lysine residues, clearly indicating that 2 residues among IS lysine residues of ALR (Terada et al., 1985) may be involved in enzymatic activity, contrary to the results that a single lysine residue involves to the activity of ALRs from pig muscle OI chicken kidney (Davidson, 1987; Flynn et al., 1989). Though two lysine residues were

100 (A) Nucleotide 80

60

40

20 -8.5

-7.5

-6.5

-5.5

-4.5

-3.5

-2.5

-1.5

Log (Nucleotide COW.(M))

90

. (B) Substrate 80

60

-6.5

-5.5

-4.5

-3.5

-2.5

-1.5

Log (Substrate cont.(M)) Fig. 3. Protection of ALR with pyridine nucleotides and SubSkiteS as a function of concentration from TNBS-inactivation. The enzyme (40 pgg/ml) was incubated for 120 min at 25 C with 0.3 mM TNBS in the presence of various concentrations of cofactors (A) or substrates (B). 0. p-NADPH: A, fi-NADP+; m, /S-NADH; +, P-NAD+; 0, p-carboxybenzaldehyde (pCBA); 0. u,L-glyceraldehyde; a, u-glucuronate.

464

Tomoyuki Terada

20 r =0.984

“0.0

1.0 2.0 Modified lysine residues (mol/mol Enz.)

Fig. 4. The number of modified lysine residues of ALR during the inactivation of ALR with TNBS. The enzyme (SOO~g/ml) was incubated with 0.3 mM TNBS and then aliquot samples were taken out for the determination of their remaining activities and the loss of lysine residues by the modification with TNBS.

modified with TNBS in the inactivated ALR, one lysine residue among these may be a residue corresponding to a residue which may be involved in 2’-adenylate moiety-binding and the other may be a unique residue for the binding of 5’-phosphate. Under a higher concentration of TNBS (over 0.5 mM) and longer incubationtime (over 120 min), ALR lost 2 or 4 lysine residues with or without /I-NADP+ (89 or 4% remaining activity, respectively), indicating that 2 lysine residues may also be modified nonspecifically and the other 2 lysine residues may be protected by fl-NADP+, though the enzyme was protected or inactivated with various nucleotides or substrates (details were not shown) the same as the modification with 0.3 mM TNBS. Additionally, the study on the inactivation and the determination of residual lysine residues during inactivation of ALR with 0.5 mM TNBS in the presence of 10 mM NAD+ showed two different results from the case of NADP(H), that is, 60min-incubated ALR

(8.5% remaining activity) was still able to be adsorbed to 2’,5’-ADP-Sepharose and 180 minincubated ALR (2.5% remaining activity) was not adsorbed completely. Furthermore, the results of absorption spectrum of ALR with /I-NADP(H) indicated a typical binary complex formation between enzyme and pyridine nucleotides. Though the same results were obtained using ALR with /?-NAD(H), the different absorption spectrum was significantly smaller than that of ALR with fi-NADP(H) (10% or less). As mentioned in the “Results” section, the inactivated enzyme (5-10% remaining activity) lost its nucleotide-binding ability significantly (data not shown). When approx. 50% inactivated enzyme was prepared with TNBS, we successfully separated a modified ALR from an unmodified ALR on an affinity column chromatography of Dye Matrex Red A or 2’,5’-ADP Sepharose, showing that the inactivated enzyme (unadsorbed fraction) lost nucleotide-binding ability in the different absorption spectrum study and about 2 lysine residues in the titration study with fluorescamine together with its activity (data not shown). In conclusion, from the inactivation studies by heat-, TNBS- and PAL-P-treatments and protection studies with various nucleotides, 2 lysine residues may play an important role in the pyridine nucleotide-binding located nearby in the nucleotide binding site of bovine liver high-K,,, aldehyde reductase. One residue among these two must be a residue to Lysz6’ and be located on the 2’-adenylate moiety binding site, and the other a residue corresponding to Lys” of human lens AR for interaction with a 5’-phosphate group (Wilson et al., 1992). Therefore, two lysine residues may play an important role in the NADP(H)-fixing on the enzyme molecule through both phosphates (2’- and 5’-)-binding.

Table 3. Residual lysine residue(s) of bovine liver high-K,,, aldehyde reductase (ALR) by the modification of trinitrobenzene sulfonate (TNBS). ALR (SOO~gjml) was incubated with 0.3 mM TNBS in the presence or absence of 1 mM P-NADP+, 1 mM b-NAD+ or IOmM D-erythrose for 120min. Modified lysine residue(s) is calculated by the determination with the fluorescamine method as described in the text. The values in this table indicate the averages of 4 experiments Remaining Modified lysine residues f SD activity i SD (mol/mol enzyme) Addition W) 96.5 + I .05 0.05 + 0.08 No addition TNBS 24.0 -+ I .98 1.87+0.16 0.17+0.13 TNBS + /I-NADP+ 98.9 + 1.66 28.1 k 2.83 1.65 f 0.28 TNBS + p-NAD+ TNBS + D-Erythrose 19.1 + 3.01 1.98 f 0.22

Lysine residues in bovine liver ALR SUMMARY

Bovine liver high-K, aldehyde reductase (ALR) was inactivated with heat-, 2,4,6-trinitrobenzene sulfonate (TNBS)- and pyridoxal Y-phosphate (PAL-P)-treatments under the following condition of 47°C 0.3 and 0.03 mM, respectively, in the pseudo-first-order kinetic manners as a function of the incubation-time. Various nucleotides, especially p -NADPH and fl-NADP+ , showed an effective protection on these ALR-inactivations. Among the nucleotides which were tested in this study, some nucleotides which have a 2’-phosphate group, showed a significant protection to these ALR(A) j3-NADPH

vs

165

inactivations. However, typical substrates for ALR, t&r.-glyceraldehyde, D-erythrose, D-glucuronate and p -carboxybenzaldehyde, could not protect the enzyme at all. The TNBSinactivation of ALR advanced with the decrease in number of lysine residues. Completely inactivated enzyme was estimated to have 2.07 TNBS-modified lysine residues/m01 enzyme by the determination of free amino group using fluorescamine (Ex = 390 nm, Em = 475 nm) and protected enzyme with fi-NADP+ (96.5% activity was remained) might lose 0.17 lysine residue/m01 enzyme. This result suggested that 2 lysine residues have an important role in the

ALR

r = 0.996

l/[NADPH/Enz.]

(B) P-NADP+

vs ALR

140 120 100 80 60 40 20 0 -0.5

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

l/INADP+/Enz.] Fig. 5. Kinetics of cofactor-binding with ALR. The increase of absorbance at 380 or 360 nm with the addition of /?-NADPH or @-NADP+ to the ALR solution (I 2.8 p M) was measured, respectively. Double reciprocal plots of the absorbances and concentrations were replotted in the figures. (A) fl-NADPH vs ALR. (B) /3-NADP+ vs ALR.

466

Tomoyuki

activity of j?-NADPH-binding. On the absorption spectrum study of ALR with P-NADPH or /?-NADP+, the absorbance at 380 or 360 nm increased with the addition of nucleotides to the enzyme solution, respectively. Kd-values for fi-NADPH or /I-NADP+ were estimated to be 0.48 or 4.7 PM, respectively. The present results suggest that lysine residues play an important role in the /I-NADP(H) binding in the ALR enzyme molecule, namely, one residue is involved in 2’-adenylate moiety-binding and the other 5’-phosphate-binding. REFERENCES Anderson B. M., Anderson C. D. and Churchich J. E. (1966) Inhibition of glutamic dehydrogenase by pyridoxal S-phosphate. Biochemistry 5, 2893-2900. BGhlen P., Stein S., Dirman W. and Udenfriend S. (1973) Fluorometric assay of proteins in the nanogram range. Arch. biochem. Biophys. 155, 213-220. Bohren K. M., Bullock B., Wermuth B. and Gabbay K. H. (1989) The aldo-keto reductase superfamily. cDNA and deduced amino acid sequences of human aldehyde and aldose reductases. J. biol. Chem. 264, 9547-9551. Bohren K. M., Page J. L., Shankar R., Henry S. P. and Gabbay K. H. (1991) Expression of human aldose and aldehyde reductases. Site-directed mutagenesis of a critical lysine. J. biol. Chem. 266, 24031-24037. Colombo G. and Marcus F. (1974) Modification of fructose l,6-diphosphatase with pyridoxal-5’-phosphate. Evidence for the participation of lysyl residues at the active site. Biochemistry 13, 3085-3091. Davidson W. S. and Flynn T. G. (1979) A functional arginine residue in NADPH-dependent aldehyde reductase from pig kidney. J. biol. Chem. 254, 3724-3729. Davidson W. S. (1987) Comparison of the active sites of aldose reductase and aldehyde reductase from chicken. Prog. clin. Biol. Res. 232, 275-285. Felsted R. L. and Bachur N. R. (1980a) Ketone reductases. Enzymat. Basis De/ox. 1, 281-293. Felsted R. L. and Bachur N. R. (1980b) Mammalian carbonyl reductases. Drug metab. Rev. 11, I-60. Flynn T. G., Gallerneault C., Ferguson D., Cromlish J. A. and Davidson W. S. (1981) Studies on the active site of pig kidney aldehyde reductase. Biochem. sot. Trans. 9, 2733275. Flynn T. G., Charington B., Lyons C., Chao H., Hyndman D. and Morjana N. (1989) Chemical modification of aldehyde and aldose reductase by pyridoxal-5’-phosphate. Prog. clin. Biol. Res. 290, 251-264. Higuchi T., Imamura Y. and Otagiri M. (1994) Chemical modification of arginine and lysine residues in coenzymebinding domain of carbonyl reductase from rabbit kidney: indomethacin affords a significant protection against inactivation of the enzyme by phenylglyoxal. Biochim. biophys. Acta 199, 81-86. lnano H. and Tamaoki B. (1986) Chemical modification of NADPH-dependent-cytochrome P-450 reductase. Presence of a lysine residue in the rat hepatic enzyme as the recognition site of 2’-phosphate moiety of the cofactor. Eur. J. Biochem. 155, 485-489. Jeffery J., Hobbs L. and Jornvall H. (1985) Glucose 6-phos-

Terada phate dehydrogenase from saccharomyces cerevisiac: characterization of a reactive lysine residue labeled with acetylsalicylic acid. Biochemistry 24, 666-67 I. Kubiseski T. J., Green N. C., Bohrhani D. W. and Flynn T. G. (1994) Studies on pig kidney aldose reductase. Identification of essential arginine in the primary and tertiary structure of the enzyme. J. biol. Chem. 269, 218332188. Levy H. M., Leber P. D. and Ryan E. M. (1963) Inactivation of myosin by 2,4-dinitrophenol and protection by adenosine triphosphate and other phosphate compounds. J. biol. Chem. 238, 3654-3659. Milhausen M. and Levy H. R. (1975) Evidence for an essential lysine in glucose 6-phosphate dehydrogenase. Eur. J. Biorhem. SO, 453-461, Mizoguchi T., Nanjo H., Umemura T., Nishinaka T., Iwata C., Imanishi T., Tanaka T., Terada T. and Nishihara T. (I 992) A novel dihydrodiol dehydrogenase in bovine liver cytosol: purification and characterization of multiple forms of dihydrodiol dehydrogenase. J. Biochem. (Tokyo) 112, 523-529. Morepeth F. F. and Dickinson F. M. (1981) Some properties of pig kidney cortex aldehyde reductase. Biochem. J. 191, 619-626. Morjana N. A., Lyons C. and Flynn T. G. (1989) Aldose reductase from human psoas muscle. Affinity labeling of an active site lysine by pyridoxal 5’-phosphate and pyridodxal 5’-diphospho-5’-adenosine. J. biol. Chem. 264, 2912-2919. Nanjo H., Terada T., Umemura T., Nishinaka T., Mizoguchi T. and Nishihara T. (1992) Characterization of bovine liver cytosolic 3a-hydroxysteroid dehydrogenase and its aldo-keto reductase activity. Int. J. Biochem. 24, 8 155820. Nanjo H., Nishinaka T., Nagai M., Terada T., Mizoguchi T. and Nishihara T. (1993) Unique dihydrodiol dehydrogenase of bovine liver: Inhibition studies and comparison with aldo/keto reductase. Adv. exp. Med. Biol. 328, 371-377. Penning T. M., Abrams W. R. and Pawlowski J. E. (1991) Affinity labeling of 3,x-hydroxysteroid dehydrogenase with 3a-bromoacetoxyandrosterone and I Ia-bromoacetoxyprogesterone. J. biol. Chem. 266, 8826-8834. Phillips N. F. B., Gross N. H. and Wood H. G. (1983) Modification of pyruvate phosphate dikinase with pyridoxal 5’-phosphate: evidence for a catalytically critical lysine residue. Biochemisfry 22, 2518-2523. Poulsom R. (1986) Inhibition of hexonate dehydrogenase and aldose reductase from bovine retina by sorbinil, Statil and valproate. Biochem. Pharmacol. 35, 2955-2959. Ranch S., Zapponi M. C. and Ferri G. (1969) Inhibition of pyridoxal-phosphate of glyceraldehyde-3-phosphate dehydrogenase. Eur. J. Biochem. 8, 3255331. Rondeau J. M., Ttte-Favier F., Podjarny A., Reymann J.-M., Barth P., Biellmann J.-F. and Moras D. (1992) Novel NADPH-binding domain revealed by the crystal structure of aldose reductase. Nature 355, 469-472. Terada T., Kohno T., Samejima T., Hosomi S., Mizoguchi T. and Uehara K. (1985) Purification and properties of beef liver aldehyde reductase catalyzing the reduction of o-erythrose 4-phosphate. J. Biochem. (Tokyo) 97, 79-87. Terada T., Nanjo H., Shinagawa K.. Umemura T., Nishinaka T., Mizoguchi T. and Nishihara T. (1993) Modulation of 3a-hydroxysteroid dehydrogenase activity by the redox state of glutathione. J. Enzyme. Inhib. 7, 33-41.

Lysine residues in bovine liver ALR Turner A. J. and Flynn T. G. (1982) The nomenclature of aldehyde reductases. bog. c/in. Biol. Res. 114, 401-402. Vogel K., Platt K.-L. and Oesch F. (1982) Dihydrodiol dehydrogenase: Substrate specificity, inducibility and tissue distribution. Arch. Toxicol. 5, 360-364. Wermuth B. and Munch J. D. B. (1979) Reduction of biogenic aldehyde by aldehyde reductase and alcohol dehydrogenase from human liver. Biochem. Pharmacol. 28, 1431-1433.

Wermuth B., Munch J. D. B. and von Wartburg J.-P. (1977) Purification and properties of NADPH-dependent

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aldehyde reductase from human liver. J. hiol. Chem. 253, 3821-3828. Yokoyama T., Matsuura Y., Yamashita K., Tanimoto T. and Nishimura C. (1992) Site-directed mutagenesis of His-42, His-188 and Lys-263 of human aldose reductase. Biochem.

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Wilson D. K., Bohren K. M., Gabbay K. H. and Quiocho F. A. (1992) An unlikely sugar substrate site in the 1.65 8, structure of the human aldose reductase holoenzyme implicated in diabetic complications. Science 257, 8 l-84.