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The methionine salvage pathway converts the methylthioribose moiety of 5'-(methylthio)-adenosine to methionine via a series of biochemical steps. One enzyme active in this pathway, a bifunctional enolase-phosphatase called E-1 that promotes oxidative cleavage of the synthetic substrate 2,3-diketo-1-phosphohexane to 2-keto-pentanoate, has been purified from Klebsiella pneumoniae and is characterized in the preceding paper (Myers, R., Wray, J., Fish, S., and Abeles, R. H. (1993) J. Biol. Chem. 268, 24785-24791). We synthesized degenerate oligonucleotides corresponding to portions of the amino terminus of E-1. These oligonucleotides were used as polymerase chain reaction primers on whole genomic DNA from Klebsiella oxytoca. This resulted in an 82-base pair DNA fragment that was used as a hybridization probe to obtain a clone of the E-1 gene from a K. oxytoca gene library. The DNA sequence of the E-1 coding region was determined, and the amino acid sequence of E-1 was deduced. E-1 appears to represent a novel class of enzymes since no homology to known enzymes was found. Cloning the gene from K. oxytoca on a multicopy plasmid leads to overproduction of E-1 enzyme that has properties indistinguishable from those of the enzyme from K. pneumoniae.

The 5-methylthio-D-ribose moiety of 5'-(methylthio)-adenosine is converted to methionine in a wide variety of organisms. 2,3-Diketo-5-methylthio-1-phosphopentane is an advanced intermediate in the methionine recycling pathway present in the Gram-negative bacterium Klebsiella pneumoniae. This unusual metabolite is oxidatively cleaved to yield formate (from C-1), 2-keto-4-methylthiobutyrate (the transamination product of methionine), and 3-methylthiopropionate. To further characterize this oxidative conversion, the desthio analog of the naturally occurring diketone, namely 2,3-diketo-1-phosphohexane I, was synthesized. If the metabolism of I is analogous to that of 2,3-diketo-5-methylthio-1-phosphopentane it should be converted to formate, 2-ketopentanoate, and butyrate. An enzyme (E-1), which mediates the oxidative conversion of I to formate and 2-ketopentanoate, was isolated from extracts of K. pneumoniae. E-1 was purified 100-fold to homogeneity in 10% yield. The native enzyme is a monomeric protein of M(r) 27,000. The activity of E-1 requires magnesium ion as a cofactor. No other prosthetic groups were detected. Incubation of the enzyme with I, under anaerobic conditions, led to the discovery of two intermediates. These species have been identified by 1H and 13C NMR, UV-visible spectroscopy, and model chemistry studies as 2-hydroxy-3-keto-1-phospho-1-hexene II, generated by enolization of I; and 1,2-dihydroxy-3-keto-1-hexene III, generated by enzymatic dephosphorylation of II. Intermediates II and III are released from the active site of the enzyme; III accumulates under anaerobic conditions. Under aerobic conditions, III is non-enzymically oxidized to 2-ketopentanoate, formate, and other products. Compound II was also generated by heating I at pH 7.5 for 7 min. Action of alkaline phosphatase on II produces III.

Considerable amounts of five glycolytic enzymes glucosephosphate isomerase, glyceraldehyde-phosphate dehydrogenase, aldolase, pyruvate kinase, and lactate dehydrogenase, became fixed when intact synaptosomes were incubated with glutaraldehyde. Other glycolytic enzymes were immobilized much less by this procedure. The lactate dehydrogenase isoenzymes showed a variable response to glutaraldehyde fixation. The isoenzymes enriched in muscle subunits were rapidly immobilized by glutaraldehyde, while the isoenzymes enriched in heart subunits, especially H4, were not. It is suggested that the enzymes which were immobilized are located near the synaptosomal membrane, perhaps in association with actin, which is found at this site. The enzymes that showed a much smaller degree of fixation were either randomly distributed in the synaptoplasm or less susceptible to fixation.

We have recently shown that the estrogen-induced protein of rat uterus (IP) is indistinguishable from a constitutive protein in rat brain, and that the brain type gamma gamma isozyme of enolase (EC 4.2.1.11) is a component of IP. Here we report that the brain type BB isozyme of creatine kinase (EC 2.7.3.2) is the major component of IP. The two IP components, creatine kinase BB and enolase gamma gamma, were copurified from rat brain by ammonium sulfate fractionation and DEAE-cellulose chromatography. The enzymes were separated on reactive blue 2 agarose, which absorbs creatine kinase BB but not enolase gamma gamma, at 40 mMNaCl, pH 5.2. The major component of IP was identified as the BB isozyme of creatine kinase on the basis of its specific enzyme activity, chromatographic behavior, and specific immunoprecipitation by anti-creatine kinase BB antiserum. The identity of the major component of IP and creatine kinase BB was confirmed by double isotope ratio analysis, limited protease digestion patterns, and the rapid increase in the rate of synthesis of creatine kinase in rat uterus in response to estrogen. IP has been a favored marker for estrogen action in rat uterus because of its early response to the hormone both in vivo and in vitro. The identification of the major component of IP as the BB isozyme of creatine kinase is a step toward understanding the function of IP in the early responses of the uterus to estrogen and reveals a further advantage for IP as a marker for the study of gene expression, and as a possible enzymic marker for hormone responsive tumors.