• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of jbacterPermissionsJournals.ASM.orgJournalJB ArticleJournal InfoAuthorsReviewers
J Bacteriol. Sep 1980; 143(3): 1458–1465.
PMCID: PMC294534

In situ reactivation of glycerol-inactivated coenzyme B12-dependent enzymes, glycerol dehydratase and diol dehydratase.

Abstract

The catalytic properties of coenzyme B12-dependent glycerol dehydratase and diol dehydratase were studied in situ with Klebsiella pneumoniae cells permeabilized by toluene treatment, since the in situ enzymes approximate the in vivo conditions of the enzymes more closely than enzymes in cell-free extracts or cell homogenates. Both dehydratases in situ underwent rapid "suicidal" inactivation by glycerol during catalysis, as they do in vitro. The inactivated dehydratases in situ, however, were rapidly and continually reactivated by adenosine 5'-triphosphate (ATP) and Mn2+ in the presence of free adenosylcobalamin, although in cell-free extracts or in cell homogenates they could not be reactivated at all under the same reaction conditions. ATP was partially replaced by cytidine 5'-triphosphate or guanosine 5'-triphosphate but not by the beta, gamma-methylene analog of ATP in the in situ reactivation. Mn2+ was fully replaced by Mg2+ but only partially by Co2+. Hydroxocoblamin could not replace adenosylcobalamin in reactivation mixtures. The ability to reactivate the glycerol-inactivated dehydratases in situ was only seen in cells grown anaerobically in glycerol-containing media. This suggests that some factor(s) required for in situ reactivation is subject to induction by glycerol. Of the two possible mechanisms of in situ reactivation, i.e., the regeneration of adenosylcobalamin by Co-adenosylation of the bound inactivated coenzyme moiety (B12-adenosylation mechanism) and the displacement of the bound inactivated coenzyme moiety by free adenosyl-cobalamin (B12-exchange mechanism), the former seems very unlikely from the experimental results.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (1.3M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Images in this article

Click on the image to see a larger version.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • ABELES RH, BROWNSTEIN AM, RANDLES CH. beta-Hydroxypropionaldehyde, an intermediate in the formation of 1,3-propanediol by Aerobacter aerogenes. Biochim Biophys Acta. 1960 Jul 15;41:530–531. [PubMed]
  • Bachovchin WW, Eagar RG, Jr, Moore KW, Richards JH. Mechanism of action of adenosylcobalamin: glycerol and other substrate analogues as substrates and inactivators for propanediol dehydratase--kinetics, stereospecificity, and mechanism. Biochemistry. 1977 Mar 22;16(6):1082–1092. [PubMed]
  • Bachovchin WW, Moore KW, Richards JH. Mechanism of action of adenosylcobalamin: hydrogen transfer in the inactivation of diol dehydratase by glycerol. Biochemistry. 1978 May 30;17(11):2218–2224. [PubMed]
  • Baker JJ, van der Drift C, Stadtman TC. Purification and properties of -lysine mutase, a pyridoxal phosphate and B 12 coenzyme dependent enzyme. Biochemistry. 1973 Mar 13;12(6):1054–1063. [PubMed]
  • BRADY RO, CASTANERA EG, BARKER HA. The enzymatic synthesis of cobamide coenzymes. J Biol Chem. 1962 Jul;237:2325–2332. [PubMed]
  • Forage RG, Foster MA. Resolution of the coenzyme B-12-dependent dehydratases of Klebsiella sp. and Citrobacter freundii. Biochim Biophys Acta. 1979 Aug 15;569(2):249–258. [PubMed]
  • Jackson RW, DeMoss JA. Effects of toluene on Escherichia coli. J Bacteriol. 1965 Nov;90(5):1420–1425. [PMC free article] [PubMed]
  • LEE HA, Jr, ABELES RH. Purification and properties of dioldehydrase, and enzyme requiring a cobamide coenzyme. J Biol Chem. 1963 Jul;238:2367–2373. [PubMed]
  • LOWRY OH, ROSEBROUGH NJ, FARR AL, RANDALL RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed]
  • Morley CG, Stadtman TC. Studies on the fermentation of D-alpha-lysine. Purification and properties of an adenosine triphosphate regulated B 12-coenzyme-dependent D-alpha-lysine mutase complex from Clostridium sticklandii. Biochemistry. 1970 Dec 8;9(25):4890–4900. [PubMed]
  • Pawelkiewicz J, Zagalak B. Enzymic conversion of glycerol into beta-hydroxy-propionaldehyde in a cell-free extract from Aerobacter aerogenes. Acta Biochim Pol. 1965;12(3):207–218. [PubMed]
  • PETERKOFSKY A, WEISSBACH H. Release of inorganic tripolyphosphate from adenosine triphosphate during vitamin B-12 coenzyme biosynthesis. J Biol Chem. 1963 Apr;238:1491–1497. [PubMed]
  • Poznanskaya AA, Yakusheva MI, Yakovlev VA. Study of the mechanism of action of adenosylcobalamindependent glycerol dehydratase from Aerobacter aerogenes. II. The inactivation kinetics of glycerol dehydratase complexes with adenosylobalamin and its analogs. Biochim Biophys Acta. 1977 Sep 15;484(1):236–243. [PubMed]
  • Reeves RE, Sols A. Regulation of Escherichia coli phosphofructokinase in situ. Biochem Biophys Res Commun. 1973 Jan 23;50(2):459–466. [PubMed]
  • Schneider Z, Larsen EG, Jacobson G, Johnson BC, Pawelkiewicz J. Purification and properties of glycerol dehydrase. J Biol Chem. 1970 Jul 10;245(13):3388–3396. [PubMed]
  • Schneider Z, Pawelkiewicz J. The properties of glycerol dehydratase isolated from Aerobacter aerogenes, and the properties of the apoenzyme subunits. Acta Biochim Pol. 1966;13(4):311–328. [PubMed]
  • SMILEY KL, SOBOLOV M. A cobamide-requiring glycerol dehydrase from an acrolein-forming Lactobacillus. Arch Biochem Biophys. 1962 Jun;97:538–543. [PubMed]
  • Toraya T, Fukui S. Immunochemical evidence for the difference between coenzyme-B12-dependent diol dehydratase and glycerol dehydratase. Eur J Biochem. 1977 Jun 1;76(1):285–289. [PubMed]
  • Toraya T, Honda S, Fukui S. Fermentation of 1,2-propanediol with 1,2-ethanediol by some genera of Enterobacteriaceae, involving coenzyme B12-dependent diol dehydratase. J Bacteriol. 1979 Jul;139(1):39–47. [PMC free article] [PubMed]
  • Toraya T, Honda S, Kuno S, Fukui S. Coenzyme B12-dependent diol dehydratase: regulation of apoenzyme synthesis in Klebsiella pneumoniae (Aerobacter aerogenes) ATCC 8724. J Bacteriol. 1978 Aug;135(2):726–729. [PMC free article] [PubMed]
  • Toraya T, Kuno S, Fukui S. Distribution of coenzyme B12-dependent diol dehydratase and glycerol dehydratase in selected genera of Enterobacteriaceae and Propionibacteriaceae. J Bacteriol. 1980 Mar;141(3):1439–1442. [PMC free article] [PubMed]
  • Toraya T, Shirakashi T, Kosuga T, Fukui S. Substrate specificity of coenzyme B12-dependent diol dehydrase: glycerol as both a good substrate and a potent inactivator. Biochem Biophys Res Commun. 1976 Mar 22;69(2):475–480. [PubMed]
  • Toraya T, Sugimoto Y, Tamao Y, Shimizu S, Fukui S. Propanediol dehydratase system. Role of monovalent cations in binding of vitamin B 12 coenzyme or its analogs to apoenzyme. Biochemistry. 1971 Aug 31;10(18):3475–3484. [PubMed]
  • Toraya T, Ushio K, Fukui S, Hogenkamp PC. Studies on the mechanism of the adenosylcobalamin-dependent diol dehydrase reaction by the use of analogs of the coenzyme. J Biol Chem. 1977 Feb 10;252(3):963–970. [PubMed]
  • Vitols E, Walker GA, Huennekens FM. Enzymatic conversion of vitamin B-12s to a cobamide coenzyme, alpha-(5,6-dimethylbenzimidazolyl)deoxyadenosylcobamide (adenosyl-B-12). J Biol Chem. 1966 Apr 10;241(7):1455–1461. [PubMed]
  • Weitzman PD, Hewson JK. In situ regulation of yeast citrate synthase. Absence of ATP inhibition observed in vitro. FEBS Lett. 1973 Oct 15;36(2):227–231. [PubMed]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...