J. Nutr. Sci. Vitaminol., 28, 225-236, 1982 the Mechanism of in Situ
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J. Nutr. Sci. Vitaminol.,28, 225-236, 1982 The Mechanism of In Situ Reactivation of Glycerol Inactivated Coenzyme B12-Dependent Enzymes, Glycerol Dehydratase and Diol Dehydratasel Kazutoshi USHIO, Susumu HONDA, Tetsuo TORAYA,2 and Saburo FUKUI3 Laboratory of Industrial Biochemistry, Department of Industrial Chemistry, Faculty of Engineering, Kyoto University, Yoshida, Sakyo-ku, Kyoto 606, Japan (Received September 28, 1981) Summary In the previous paper (S. Honda, T. Toraya, and S. Fukui, J. Bacteriol., 143, 1458-1465 (1980)), we reported that the glycerol inactivated holoenzymes of adenosylcobalamin-dependent glycerol dehy dratase and diol dehydratase are rapidly and continually reactivated in toluene-treated cells (in situ) by adenosine 5•L-triphosphate (ATP) and divalent metal ions in the presence of free adenosylcobalamin. To elucidate the mechanism of this in situ reactivation, the nature of the binding of various irreversible cobalamin inhibitors to the dehydratases in situ was investigated. In the presence of ATP and Mn2+, enzyme-bound hydroxocobalamin, cyanocobalamin and methylcobalamin were rapidly displaced by added adenosylcobalamin. Without ATP and Mn2+, such displacement did not take place. In contrast, enzyme-bound adeninyl butylcobalamin and adenosylethylcobalamin were essentially not displace able by the free coenzyme even in the presence of ATP and Mn2+. Inosylcobalamin was a very weak inhibitor irrespective of the presence of ATP and Mn2+ . These results indicate that the relative affinity of the enzymes in situ for the cobalamins with simple Cof ligands was markedly lowered in the presence of ATP and Mn2+, whereas that for the cobalamins with adenine-containing ligands was not. When the glycerol inactivated holoenzymes in situ were dialyzed against a buffer containing ATP and Mg2+, the inactivated coenzyme moiety dissociated from the enzymes leaving apoproteins. Kinetic evidence was also obtained with the dehydratases in situ that continual displacement of the inactivated 1 This is Paper XVIII in the series concerning "Coenzyme B 12-Dependent Dehy dratases." 2 Address correspondence to this author at the present address: Department of Chemistry, College of Liberal Arts and Sciences, Kyoto University, Yoshida, Sakyo-ku, Kyoto 606, Japan. 3 牛 尾 一 利,本 多 進, 2 虎 谷 哲 夫 ,福 井 三 郎 225 226 K. USHIOet al. coenzyme moiety by adenosylcobalamin takes place during the glycerol dehydration reaction in the presence of ATP and Mn2+. Since the adenosyl group of the bound coenzyme is irreversibly removed from the cobalamin moiety during inactivation by glycerol, all of these data constitute clear evidence that the inactivated holo-dehydratases are re activated in situ in the presence of ATP and Mn2+ by displacement of the modified coenzyme moiety by free intact adenosylcobalamin (i.e. selective B12-exchange mechanism). Key Words coenzyme B12, glycerol dehydratase, diol dehydratase, sui cide inactivation, mechanism of in situ reactivation, Toluene-treated cell Coenzyme B12 (adenosylcobalamin)-dependent glycerol dehydratase (glycerol hydro-lyase, EC 4.2.1.30) and diol dehydratase (DL-1, 2-propanediol hydro-lyase, EC 4.2.1.28) are isofunctional but immunologically different enzymes, both of which catalyze the conversion of glycerol to ƒÀ-hydroxypropionaldehyde, that of 1, 2 - propanediol to propionaldehyde and that of 1, 2-ethanediol to acetaldehyde (1-6). As described in our previous paper (7), glycerol is an important physiological substrate for glycerol dehydratase and, in some cases, for diol dehydratase as well. Recent studies in this and other laboratories, however, have indicated that both dehydratases undergo suicidal inactivation by glycerol during catalysis (2, 3, 8-10). This apparent inconsistency was eliminated by our recent finding that the glycerol inactivated dehydratases are rapidly reactivated in toluene-treated cells (in situ) by ATP and divalent metal ions in the presence of free adenosylcobalamin (11). During inactivation by glycerol, the carbon-cobalt bond of the enzyme-bound coenzyme is irreversibly broken forming 5•L-deoxyadenosine from the adenosyl group (3, 12, and T. Toraya, T. Tobimatsu, T. Kawamura, and S. Fukui, manuscript in preparation). We have shown that the apoprotein itself is not damaged during inactivation. Therefore, two possible mechanisms were considered for this reactivation in situ: the regeneration of adenosylcobalamin by cobalt adenosylation of the bound inactivated coenzyme moiety (B12-adenosylation mechanism) and the displacement of the bound inactivated coenzyme moiety by free adenosylcobalamin (B12-exchange mechanism). The experimental results reported in our previous paper indicated that the B12-adenosylation mechanism is very unlikely (11). In this paper, we report definite evidence that the glycerol-inactivated glycerol and diol dehydratases are reactivated in situ through the B12-exchange mechanism. MATERIALS AND METHODS Chemicals. Crystalline adenosylcobalamin and methylcobalamin were gifts from Kyowa Hakko Kogyo Co., Ltd., Tokyo and Eisai Co., Ltd., Tokyo, respec tively. Adeninylbutylcobalamin, adenosylethylcobalamin, and inosylcobalamin were kindly supplied by Professor H. P. C. Hogenkamp, University of Minnesota, J. Nutr. Sci. Vitaminol. REACTIVATION OF B12 ENZYMES 227 Minneapolis, Minnesota. Cyanocobalamin was obtained from Glaxo Laboratories Ltd., Greenford, England. Hydroxocobalamin was prepared by aerobic photolysis of methylcobalamin (13, 14). All other chemicals were reagent-grade commercial products, and were used without further purification. Bacteria and growth. As described in the previous paper (11), glycerol-grown cells of Klebsiella pneumoniae ATCC 25955 (formerly Aerobacter aerogenes PZH 572, Warsaw) and glycerol-1, 2-propanediol-grown cells of K. pneumoniae ATCC 8724 (formerly A. aerogenes) were used as the sources of glycerol dehydratase and diol dehydratase, respectively (8, 15). The bacteria were grown at 30•Ž or 37•Ž without aeration in a complex medium containing 5.4g of KH2PO4, 1.2g of (NH4)2SO4, 0.4g of MgSO4 7H2O, 2.0g of yeast extract, 2.0g of tryptone, and 9.2g of glycerol (glycerol medium) or 9.2g of glycerol plus 5.7g of 1, 2-propanediol (glycerol-1, 2-propanediol medium) in 1 liter of tap water (7, 8, 15). The medium was adjusted to pH 7.1 with KOH. Permeabilized cells. Bacterial cells were permeabilized by toluene treatment, as described before (11). The concentration of the bacteria and toluene-treated cells was determined by comparison of the turbidity of the suspensions at 580nm with standard curves (11). Purification of diol dehydratase. The purified preparation of the diol dehy dratase of K. pneumoniae ATCC 8724 was obtained as described before (16) and used for the kinetic measurements as an in vitro system. Enzyme assays. The activities of the glycerol dehydratase and diol dehydratase in vitro and in situ were determined by the 3-methyl-2-benzothiazolinone hydrazone method of Toraya et al. (8, 17), as described in the previous paper (11). RESULTS AND DISCUSSION Effect of hydroxocobalamin on reactivation in situ As reported previously (11), hydroxocobalamin itself is not able to replace adenosylcobalamin in the reactivation mixture for the glycerol-inactivated glycerol dehydratase in situ. When both adenosylcobalamin and hydroxocobalamin were added together in equimolar quantities (15ƒÊM each), the reactivation was 46 inhibited (Table 1). It should be noted that this percentage is much smaller than that of inhibition of the dehydratase reaction obtained with hydroxocobalamin without ATP and Mn2+ (>90%). This result suggested that the relative affinity of the in situ enzyme for hydroxocobalamin was greatly lowered by the presence of ATP and Mn2+. Displacement of the enzyme-bound cobalamin inhibitors in situ by adenosylcobalamin The result described above led us to examine reactivation of the inactive model complexes of the dehydratases with cyanocobalamin, hydroxocobalamin, methyl cobalamin, adeninylbutylcobalamin, and adenosylethylcobalamin. These model complexes formed in situ were incubated in the reactivation mixture, namely with Vol.28, No. 3, 1982 228 K. USHIO et al. Table 1. Effect of hydroxocobalamin on the reactivation in situ of glycerol-inactivated glycerol dehydratase. Toluene-treated cells of K. pneumoniae ATCC 25955 were incubated at 37•Ž for 30min in a mixture containing 15ƒÊM adenosylcobalamin, 0.2M glycerol, 0.05M KCl and 0.04M potassium phosphate buffer (pH 8.0). Free adenosyl cobalamin and glycerol were removed by washing and dialysis of the cells against 0.05M potassium phosphate buffer (pH 8.0). The experiment for reactivation in situ was carried out at 37•Ž for 10min with 0.009mg (dry weight) of the inactivated cells in the reactivation mixture. The complete system contained 0.2M glycerol, 0.05M KCl, 0.03M potassium phosphate buffer (pH 8.0), 15ƒÊM adenosylcobalamin, 3mM ATP, 3mM MnCl2 and inactivated cells, in a total volume of 1.0ml. Fig. 1. Time course of displacement in situ of the glycerol dehydratase-bound coba lamin inhibitors by adenosylcobalamin. Toluene-treated cells (0.022-0.025mg of dry cells) of K. pneumoniae ATCC 25955 were incubated at 37•Ž in a mixture containing 0.2 mmol of 1, 2-propanediol, 0.05 mmol of KCl, 0.035 mmol of potassium phosphate buffer (pH 8.0) and 15 nmol of hydroxocobalamin (A), 15 nmol of cyanocobalamin (B), or 5 nmol of adeninylbutylcobalamin (C), with (•¤) or without (_??_) 3ƒÊmol of ATP and 3ƒÊmol of MnCl2, in a total volume of 0.9ml. Adenosylcobalamin (15 nmol) was added to