Selective inactivation of rabbit muscle glycerophosphate dehydrogenase by diazotized 3-aminopyridine adenine dinucleotide

ARCHIVES OF BIOCHEMISTRY Vol.

188, No. 1, May,

Selective Dehydrogenase BRUCE

Department

ANU BIWHYSKS pp. 214-219, 1978

Inactivation of Rabbit Muscle by Diazotized 3-Aminopyridine

M. ANDERSON,’

of Biochemistry

and Nutrition,

Glycerophosphate Adenine Dinucleotide’

SUSAN T. KOHLER, ANDERSON Virginia Polytechnic Virginia 24061 Received

January

Institute

AND

CONSTANCE

and State

Unioersity,

D.

BZackshurg,

31, 1978

Rabbit muscle glycerophosphate dehydrogenase was rapidly and irreversibly inactivated at pH 7.0 and 4°C by low concentrations of diazotized 3-aminopyridine adenine dinucleotide. The enzyme was protected from inactivation by the presence of NAD. During the inactivation process, 2 mol of diazotized 3-aminopyridine adenine dinucleotide was covalently attached per 1 mol of enzyme or one diazotized 3-aminopyridine adenine dinucleotide per active site. The selective modification of an active-site cysteine residue was indicated by the observation that one sulfhydryi group per active site was lost during the inactivation process. Diazotized 3-aminopyridine adenine dinucleotide did not irreversibly inactivate bovine lactate dehydrogenase, Mq isozyme, or bovine heart mitochondrial malate dehydrogenase, presumably due to the inaccessibility of active-site sulfhydryl groups. Diazotized 3aminopyridine adenine dinucleotide was selectively bound to the lactate dehydrogenase as a coenzyme-competitive inhibitor.

In recent studies (1) the chemical conversion of NAD to 3-aminopyridine adenine dinucleotide (AAD) through the Hoffman hypobromite reaction was described. The product AAD could be diazotized by reaction with nitrous acid and the resulting diazonium chloride was azocoupled with arylamines to form azodyes. Further studies (2) of diazotized AAD demonstrated the compound to be a site-labeling reagent for yeast alcohol dehydrogenase. In these studies diazotized AAD was observed to inactivate the dehydrogenase rapidly by covalently modifying one sulfhydryl group per active site of the enzyme. The selectivity of the inactivation of yeast alcohol dehydrogenase by diazotized AAD, in addition to the obvious structural analogy to the coenzyme, appears to be related to the accessibility of a sulfhydryl group at or nearby the

coenzyme binding site. Diazotized AAD reacts rapidly with a number of sulfhydryl compounds at neutral pH (2); however, azocoupling to arylamines and phenols does not appear to occur at pH’s above 3, presumably due to the formation of the diazohydroxide or diazotate. In this respect, a site-specific inactivation of dehydrogenases by diazotized AAD may require the presence of a free sulfhydryl group at the active sites of the enzymes. In the absence of such a group, reversible coenzyme-competitive inhibition by diazotized AAD might be expected. Recent studies (3) of diazotized 3aminopyridine adenine dinucleotide phosphate (AADP) with yeast glucose 6-phosphate dehydrogenase and yeast 6-phosphogluconate dehydrogenase yielded exactly these results. Previous studies (4,5) of the inactivation of rabbit muscle glycerophosphate dehydrogenase by N-alkyhnaleimides and studies of the fluorescence titration of the enzyme with NADH suggested the presence of an essential sulfhydryl group at or nearby the coenzyme binding site. It was therefore of interest to investigate the application of

’ This work was supported by Research Grant PCM 76 14720 from the National Science Foundation. ’ To whom inquiries should be addressed. 3 Abbreviations used: AAD, 3-aminopyridine adenine dinucleotide; AADP, 3-aminopyridine adenine dinucleotide phosphate; DTNB, 5,5’-dithiobis(2-nitrobenzoic acid). 214 0003~9R61/78/1881-0214$02.00/O CopyrighL 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

RABBIT

MUSCLE

GLYCERGPHOSPHATE

diazotized AAD as a site-labeling reagent for this enzyme, as well as for other dehydrogenases known to contain reactive sulfhydryl groups. MATERIALS

AND

METHODS

Materials. NAD, crystalline beef heart lactate dehydrogenase H, isozyme (EC 1.1.1.27), r,-malic acid, I,-cy-glycerophosphate, crystalline rabbit muscle ~.-aglycerophosphate dehydrogenase (EC 1.1.1.8), and DTNB were obtained from Sigma Chemical Co. Crystalline beef muscle lactate dehydrogenase Mq isozyme (EC 1.1.1.27) was obtained from Boehringer Mannheim Corp. Bovine heart mitochondrial malate dehydrogenase (EC 1.1.1.37) was prepared according to Gregory (6) and generously supplied by Dr. E. M. Gregory. AAD was prepared through the Hoffman hypobromite reaction as described by Fisher et al. (1). AADP was prepared through the NADase pyridine base exchange reaction according to Anderson et al. (3). Methods. The catalytic activity of the beef lactate dehydrogenase isozymes was determined at 25’C in 3ml reaction mixtures containing 0.05 M Tris-HCl buffer, pH 8.1, 0.2 M lithium lactate, and 30 PM NAD. The activity of bovine mitochondrial malate dehydrogenase was determined at 25°C in 3-ml reaction mixtures containing 66 mM sodium pyrophosphate buffer, pH 9.0, 7 mM malate, and 1 mM NAD. The catalytic activity of glycerophosphate dehydrogenase was determined at 25°C in 3-ml reaction mixtures containing 0.06 M Tris-HCl buffer, pH 7.86, 1.8 mM NAD, and 1.7 mM glycerophosphate. In each case, reactions were initiated by the addition of enzyme and the formation of NADH was followed spectrophotometrically at 340 nm using a Beckman Acta MVI recording spectrophotometer. All pH measurements were made using a Radiometer PHM-52 pH meter equipped with a Radiometer GK 2321-C combination electrode. DTNB titrations were carried in 4 M guanidine hydrochloride according to Ellman (7). RESULTS

Diazotized AAD was prepared at 0 to 4°C as described previously (1) by constructing the following reaction mixture. To a 0.2-ml solution of AAD (concentrations varied) was added 0.05 ml of 1 N HCl and then 0.1 ml of 0.1 M NaN02. After 10 min, 0.1 ml of 0.2 M ammonium sulfamate was added with stirring to destroy excess HN02. After an additional 10 min, 0.05 ml of a NaOH solution was added to adjust the reaction mixture to the desired pH. The exact concentration of the NaOH solution depended upon the pH required. An additional 0.4 ml

DEHYDROGENASE

INACTIVATION

215

of an appropriate buffer was then added to maintain the desired pH. To initiate the enzyme inactivation studies, which were carried out at 0 to 4°C sufficient enzyme in 0.1 ml was added to the reaction mixture and O.l-ml aliquots were removed at timed intervals and assayed for catalytic activity as described under Methods. Enzyme assays were performed at 25’C. For studies of the inactivation of beef lactate dehydrogenase Md isozyme, the diazotized AAD reaction mixture was constructed using 0.8 mM AAD, 1 N NaOH for pH adjustment, and 0.1 M potassium phosphate buffer, pH 7.6 to maintain the reaction pH. After the addition of 4 pg of enzyme, aliquots were removed from the reaction mixture every 10 min for 1 h and assayed for enzyme activity. No loss in enzyme activity was observed over this 60min period. Identical reaction mixtures containing beef lactate dehydrogenase Hq isozyme yielded the same results in that the catalytic activity of this isozyme was likewise not decreased during the 60-min incubation period. Inactivation of the Mq isozyme was also studied at pH 6.0 and again no loss in enzyme activity was observed over the 60-min incubation period. Inactivation of bovine mitochondrial malate dehydrogenase was investigated at four different pH’s at a concentration of 1 mM diazotized AAD. The reaction mixtures were adjusted to the desired pH with different concentrations of NaOH and the buffer additions were 0.1 M acetate buffer at pH 5 and 5.5 and 0.1 M sodium phosphate buffer at pH 6 and 6.5. Reactions were initiated with the addition of 40 pugof enzyme. At each pH studied, no loss in catalytic activity was observed during a 60-min incubation period. In the studies of beef lactate dehydrogenase Mq isozyme incubated with diazotized AAD, zero time activity measurements were always slightly lower than expected for the amount of enzyme being transferred to the assay mixture. This suggested the possibility that diazotized AAD was functioning as a reversible inhibitor in this case and further studies proved successful in supporting this suggestion. The inhibition by diazotized AAD was studied at 25°C in

216

ANDERSON,

KOHLER,

AND

ANDERSON

3-ml reaction mixtures containing 0.05 M Tris-HCl buffer, pH 8.1, 0.2 M lithium lactate, 0.4 pg of enzyme, inhibitor, and NAD varied from 20 to 107 PM. Effective inhibition was observed at 0.28 and 0.56 InM diazotized AAD and, as shown in Fig. 1, the inhibition was competitive with respect to NAD. The K, value calculated from these data was 0.28 mM. Although diazotized AAD was observed not to inactivate the malate and lactate dehydrogenases, it was observed to modify selectively rabbit muscle glycerophosphate dehydrogenase. For studies of the selective inactivation of glycerophosphate dehydrogenase, diazotized AAD reaction mixtures were prepared as described above using 1 N NaOH for pH adjustment and 0.1 M sodium pyrophosphate buffer, pH 7.0 to maintain the reaction pH. The concentration of diazotized AAD was varied from 63 to 420 PM and reactions were initiated with the addition of 400 pg of enzyme. Aliquots (0.1 ml) of the reaction mixture were removed at timed intervals and assayed for enzyme activity as described under Methods. The enzyme was observed to be inactivated rapidly in the presence of the diazotized AAD. No loss in catalytic activity

was observed if AAD was not present in the reaction mixture described above. Effective inactivation of glycerophosphate dehydrogenase was achieved at concentrations of diazotized AAD as low as 63 ,uM. The rate of inactivation of the enzyme increased with increasing concentrations of diazotized AAD, as shown in Fig. 2. The inactivation of the enzyme followed pseudo-first-order kinetics and observed first-order rate constants were calculated from the relationship, kohs = 0.693/th. The rate of inactivation of the enzyme was shown to be proportional to the concentration of diazotized AAD (Fig. 3). The enzyme was observed to be protected from inactivation by diazotized AAD by the presence of the natural coenzyme, NAD. Inactivation of the enzyme with 0.21 mM diazotized AAD proceeded with a ti of 5.2 min (Fig. 4, line 1). In the presence of 2.43 PM NAD the rate of inactivation was reduced to a t: of 9.8 min (Fig. 4, line 2). At a concentration of 4.86 PM NAD the rate of inactivation was further reduced to a tl of 24.5 min (Fig. 4, line 3). Inactivation of glycerophosphate dehy-

FIG. 1. Competitive inhibition of beef lactate dehydrogenase by diazotized AAD. NAD concentrations were varied from 20 to 107 pM. Reaction mixtures contained 0.05 M Tris-HCl buffer, pH 8.1,0.2 M lithium lactate, 0.4 pg of enzyme, and NAD and inhibitor as indicated in a total volume of 3.0 ml. Line 1, no inhibitor; line 2, 0.28 m&r diazotized AAD; line 3, 0.56 mM diazotized AAD.

FIG. 2. Time-dependent inactivation of rabbit muscle glycerophosphate dehydrogenase by diazotized AAD. The reaction mixtures contained 0.1 M sodium pyrophosphate buffer, pH 7.0, 400 gg of enzyme, and diazotized AAD in a total volume of 2.0 ml. The concentrations of diazotized AAD used were 125 FM (line I), 190 pM (line 2), 250 pM (line 3), and 330 PM (line 4).

RABBIT

MUSCLE

GLYCEROPHOSPHATE

FIG. 3. The effect of diazotized AAD concentration on the pseudo-first-order rate constants of glycerophosphate dehydrogenase inactivation. Reaction conditions were those described in Fig. 2. T

-1

FIG. 4. NAD protection of glycerophosphate dehydrogenase inactivation by diazotized AAD. Reaction mixtures contained 0.1 M sodium pyrophosphate buffer, pH 7.0,0.21 mM diazotized AAD, and 400 1.18of enzyme in a total volume of 2.0 ml. The concentration of NAD was zero (line l), 2.43 pM (line 2). and 4.86 PM (line 3).

drogenase by 0.18 mM diazotized AAD was studied further in reaction mixtures utilizing both Tris-HCl and sodium phosphate buffers at different pH’s in the pH range from 6 to 9. No significant differences were observed in the rates of inactivation in this pH range.

DEHYDROGENASE

217

INACTIVATION

In order to investigate in greater detail the inactivation of glycerophosphate dehydrogenase, 2 mg of the enzyme was reacted with 1.28 mM diazotized AAD in a total volume of 2 ml. Inactivation was completed within 10 min and the reaction mixture was applied to a Sephadex G-25 column (1.2 x 47 cm). A second passage through the column was required to separate completely the modified enzyme from the reagent mixture. Fractions containing the inactivated enzyme were pooled and the spectral properties of this preparation were compared to native enzyme exposed to a diazotization reaction mixture lacking AAD and processed through a Sephadex G-25 column in an identical fashion. The modified enzyme exhibited additional absorption at 260 nm not observed with the native and catalytically active enzyme. .IJsing the extinction coefficient of diazotized AAD at 260 nm (1) the number of micromoles of diazotized AAD covalently attached per micromole of enzyme were calculated. In four independent experiments, an average value of 1.81 pmol of adenyl residues/pmol of enzyme was determined to be present. The calculated values are listed in Table I. During this inactivation process, free sulfhydryl groups of the enzyme were monitored by DTNB titration (7). In three separate experiments, an average of 2.12 sullhydryl groups per molecule of enzyme were modified upon complete inactivation (Table I). TABLE I COMPAHISONOFSPECTRALANALYSISANL) SULFHYDRYL MODIFICATIONOF GLYCEROPHOSPHATEDEHYDROGENASE INACTIVATEDBY DIAZOTIZED 3-Ah4rNownrr)rNn ADENINE DINUCLEOTIDE Experiment 1 2 3 4

Moles of adenyl residues per mole of enzyme”

Moles of sullhydry1 group moditied per mole of enzymeh

1.77 1.87 1.81 1.78

2.12 2.20 2.06

n Calculated from difference spectra of native versus inactivated enzyme. ’ Calculated from DTNB titration studies. ’ DTNB titration not performed on this sample.

218

ANDERSON,

KOHLER,

DISCUSSION

Rabbit muscle glycerophosphate dehydrogenase was observed to be rapidly inactivated by low concentrations of diazotized 3-aminopyridine adenine dinucleotide (AAD). The inactivation reaction proceeded under very mild reaction conditions of neutral pH and 4°C with measurable rates of inactivation obtained with concentrations of diazotized AAD as low as 63 pM. The rabbit muscle enzyme could be effectively protected from inactivation by micromolar concentrations of NAD (Fig. 4). The pseudo-first-order rate constant for inactivation with 0.21 mM diazotized AAD was calculated to be 0.133 min-‘. At this concentration of diazotized AAD, protection by NAD was observed to be 47% at 2.43 PM and 79% at 4.86 PM. These concentrations of coenzyme are below the K,,, value of 16 pM measured under these conditions of temperature and pH. The effective protection by coenzyme suggested the possibility of a site-directed process for the inactivation reaction. This possibility was further studied by investigating the functional group involvement and the stoichiometry of the inactivation reaction. In previous studies of diazotized AAD with yeast alcohol dehydrogenase (2), the site-directed inactivation of this dehydrogenase was shown to involve the modification of one sulfhydryl group per catalytic subunit of the enzyme, which totally accounted for the number of AAD residues covalently attached to the enzyme during inactivation. Sulfhydryl modification was also involved in the selective inactivation of Neurospora nitrate reductase by diazotized AADP (8). These observations coupled with the known sensitivity of glycerophosphate dehydrogenase to sulfhydryl reagents prompted the present investigation of sulfhydryl group modification of this enzyme. Spectrophotometric analysis of glycerophosphate dehydrogenase inactivated by diazotized AAD indicated (Table I) the covalent attachment of two AAD residues per mole of enzyme (78,000 M,) or one residue per catalytic subunit. The fact that two sulfhydryl groups were modified during the inactivation process as measured by DTNB titration ruled out random modifi-

AND

ANDERSON

cation of other functional groups of the enzyme. These observations are consistent with earlier studies of the dehydrogenase which demonstrated the binding of 2 mol of coenzyme/mol of enzyme and the presence of two identical subunits per mole of enzyme (5, 9, 10). The selectivity of the inactivation of glycerophosphate dehydrogenase by diazotized AAD supports previous studies (4), indicating the presence of an essential sulfhydryl group at or nearby the coenzyme binding sites of this enzyme. The absence of a selective inactivation of beef lactate dehydrogenase Mq isozyme is an important observation with respect to the functioning of diazotized AAD as a sitelabeling reagent for dehydrogenases. Coenzyme-competitive inhibition of this lactate dehydrogenase isozyme by diazotized AAD (Fig. 1) indicated effective binding of the reagent at the coenzyme binding site and the lack of irreversible inactivation points to the absence of a reactive sulfhydryl group in the vicinity of the coenzyme binding region of the enzyme. These observations are consistent with previous studies (11) in which N-alkylmaleimide inactivation of the M, isozyme was not prevented by the presence of high concentrations of either oxidized or reduced forms of the coenzyme. These results are in obvious accord with X-ray crystallographic studies indicating that the reactive sulfhydryl groups of lactate dehydrogenase are not located in the catalytic sites of the enzyme (12). A similar observation was recently made in studies of diazotized AADP, in which the diazotized dinucleotide served as an effective coenzyme-competitive inhibitor of yeast glucose 6-phosphate dehydrogenase and yeast 6-phosphogluconate dehydrogenase but no irreversible inactivation of these enzymes was observed (3). The apparent inability of the diazotized dinucleotides to azocouple with amino acid residues such as histidine, tryptophan, and tyrosine at neutral pH suggest these compounds to be efficient probes for cysteine residues at the active site of pyridine nucleotide-dependent enzymes. REFERENCES 1. FISHER,

T. L., VERCEI~LOTTI,

S. V., AND ANDER-

RABBIT

MUSCLE

GLYCEROPHOSPHATE

SON, B. M. (1973) J. Biol. Chem. 248,4293-4299. 2. CHAN, J. K., AND ANDERSON, B. M. (1975) J. Biol. Chem. 250,67-72. 3. ANDERSON, B. M., YUAN, J. H., AND VERCF,I,LOTTI, S. V. (1975) Mol. Cell. Biochem. 8,89-96. 4. ANDERSON, B. M., KIM, S. J., AND WANG, C. N. (1970) Arch. Biochem. Biophys. 138,66-72. 5. KIM, S. J., AND ANDERSON, B. M. (1969) J. Biol. Chem. 244, 1547-1551. 6. GREGORY, E. M. (1975) J. Biol. Chem. 250, 5470-5474. 7. ELLMAN, G. L. (1959) Arch. Biochem. Biophys.

DEHYDROGENASE

INACTIVATION

219

82, 70-77. 8. AMY, N. K., GARRETT, R. H., AND ANDERSON, B. M. (1977) Biochim. Biophys. Acta 480, 83-95. 9. PFI,EIDERER, G., AND AURICCHIO, F. (1964) Biothem. Biophys. Res. Commun. 16.53-59. 10. VAN EYS, J., JUDD, J., FORD, J., AND WOMACK, W. B. (1964) Biochemistry 3, 1755-1763. 11. ANDERSON, B. M., VEHCEI,LOTTI, S. V., AND FISHER, T. L. (1976) Biochim. Biophys. Acta 350, 135-140. 12. HESS, G. P., ANDRUPLEY, J. A. (1971) Annu. Rev. Biochem. 40, 1036-1038.