Two dioxygenases (ARD and ARD') were cloned from Klebsiella pneumoniae that catalyze different oxidative decomposition reactions of an advanced aci-reductone intermediate, CH(3)SCH(2)CH(2)COCH(OH)=CH(OH) (I), in the methionine salvage pathway. The two enzymes are remarkable in that they have the same polypeptide sequence but bind different metal ions (Ni(2+) and Fe(2+), respectively). ARD converts I to CH(3)SCH(2)CH(2)COOH, CO, and HCOOH. ARD' converts I to CH(3)SCH(2)CH(2)COCOOH and HCOOH. Kinetic analyses suggest that both ARD and ARD' have ordered sequential mechanisms. A model substrate (II), a dethio analogue of I, binds to the enzyme first as evidenced by its lambda(max) red shift upon binding. The dianion formation from II causes the same lambda(max) red shift, suggesting that II bind to the enzyme as a dianion. The electron-rich II dianion likely reacts with O(2) to form a peroxide anion intermediate. Previous (18)O(2) and (14)C tracer experiments established that ARD incorporates (18)O(2) into C(1) and C(3) of II and C(2) is released as CO. ARD' incorporates (18)O(2) into C(1) and C(2) of II. The product distribution seems to necessitate the formation of a five-membered cyclic peroxide intermediate for ARD and a four-membered cyclic peroxide intermediate for ARD'. A model chemical reaction demonstrates the chemical and kinetic competency of the proposed five-membered cyclic peroxide intermediate. The breakdown of the four-membered and five-membered cyclic peroxide intermediates gives the ARD' and ARD products, respectively. The nature of the metal ion appears to dictate the attack site of the peroxide anion and, consequently, the different cyclic peroxide intermediates and the different oxidative cleavages of II. A cyclopropyl substrate analogue inactivates both enzymes after multiple turnovers, providing evidence that a radical mechanism may be involved in the formation of the peroxide anion intermediate.
The use of fluoro ketones as inhibitors of hydrolytic enzymes has been investigated. The acetylcholine analogues 6,6-dimethyl-1,1,1-trifluoro-2-heptanone and 3,3-difluoro-6,6-dimethyl-2-heptanone are inhibitors of acetylcholinesterase with Ki values of 16 X 10(-9) M and 1.6 X 10(-9) M, respectively. These fluoro ketones are 10(4)-10(5) times better as inhibitors than the corresponding methyl ketone. Since nucleophiles readily add to fluoro ketones, it is likely that these compounds inhibit acetylcholinesterase by formation of a stable hemiketal with the active-site serine residue. Fluoro ketone substrate analogues are also inhibitors of zinc metallo- and aspartylproteases. 2-Benzyl-4-oxo-5,5,5-trifluoropentanoic acid is an inhibitor of carboxypeptidase A (Ki = 2 X 10(-7) M). Trifluoromethyl ketone dipeptide analogues are good inhibitors of angiotensin converting enzyme. An analogue of pepstatin that contains a difluorostatone residue in place of statine has been prepared and found to be an extremely potent inhibitor of pepsin (Ki = 6 X 10(-11) M). The hydrated ketones are probably the inhibitory species since they are structural mimics of the tetrahedral intermediate that forms during the hydrolysis of peptide substrates.
We have synthesized peptidyl fluoromethyl ketones that are specific inhibitors of the serine proteases alpha-chymotrypsin and porcine pancreatic elastase. By analogy with the corresponding aldehydes it is assumed that the fluoromethyl ketones react with the gamma-OH group of the active site serine to form a stable hemiacetal [Lowe, G., & Nurse, D. (1977) J. Chem. Soc., Chem. Commun., 815; Chen, R., Gorenstein, D.G., Kennedy, W.P., Lowe, G., Nurse, D., & Schultz, R.M. (1979) Biochemistry 18, 921; Shah, D.O., Lai, K., & Gorenstein, D.G. (1984) J. Am. Chem. Soc. 106, 4272]. 19F NMR studies of the chymotrypsin-bound trifluoromethyl ketone inhibitors Ac-Leu-ambo-Phe-CF3 and Ac-ambo-Phe-CF3 clearly indicate that the carbonyl carbon is tetrahedral at the active site of the enzyme. The inhibitor is bound as either the stable hydrate or the hemiacetal, involving the active site serine. The effect of varying the number of amino acid residues in the peptidyl portion of the inhibitor and the number of fluorines in the fluoromethyl ketone moiety is examined. In the series of trifluoromethyl ketone elastase inhibitors, the lowering of Ki concomitant with the change from a dipeptide analogue to a tetrapeptide analogue (Ac-Pro-ambo-Ala-CF3, Ki = 3 X 10(-3) M; Ac-Ala-Ala-Pro-ambo-Ala-CF3, Ki = 0.34 X 10(-6) M) correlates well with the variation in V/K for hydrolysis of the corresponding amide substrates. This trend is indicative of the inhibitors acting as transition-state analogues [Bartlett, P.A.,& Marlowe, C.K. (1983) Biochemistry 22, 4618; Thompson, R.C. (1973) Biochemistry 12, 47].(ABSTRACT TRUNCATED AT 250 WORDS)
A dipeptidyl trifluoromethyl ketone, N-acetyl-L-leucyl-L-[1-13C]phenylalanyl trifluoromethyl ketone, was synthesized. This compound inhibits chymotrypsin with Ki = 1.2 microM [Imperiali B., & Abeles, R.H. (1986) Biochemistry 25, 3760-3767]. The complex formed between this inhibitor and alpha-chymotrypsin was examined with 1H, 13C, and 19F NMR spectroscopy to establish its structure in solution. The keto group of the trifluoro ketone is present as an ionized hemiketal group as deduced from the comparison of its 13C chemical shift with those of model hemiketals. The pKa of the hemiketal hydroxyl in the complex is approximately 4.9, which is about 4.2 units lower than the pKa of model hemiketals. This observation provides direct evidence that serine proteases are able to stabilize the oxyanions of tetrahedral adducts. Evidence is also presented for the presence of an Asp-His H bond and protonation of the imidazole group of His-57 in the tetrahedral adduct. The pKa of His-57 is higher than 10. This observation directly indicates that the pKa of His-57 is elevated in a complex containing a tetrahedral adduct.
Two enzymes, designated, E-2 and E-2, catalyze different oxidation reactions of an aci-reductone intermediate in the methionine salvage pathway. E-2 and E-2, overproduced in Escherichia coli from the same gene, have the same protein component. E-2 and E-2 are separable on an anion exchange column or a hydrophobic column. Their distinct catalytic and chromatographic properties result from binding different metals. The apo-enzyme, obtained after metal is removed from either enzyme, is catalytically inactive. Addition of Ni 2؉ or Co 2؉ to the apo-protein yields E-2 activity. E-2 activity is obtained when Fe 2؉ is added. Production in intact E. coli of E-2 and E-2 depends on the availability of the corresponding metals. These observations suggest that the metal component dictates reaction specificity.S-Methylthioadenosine, a metabolite derived from methionine (1, 2), is a strong inhibitor of polyamine biosynthesis and transmethylation reactions (2, 3). Therefore its concentration in biological systems must be tightly controlled. Control is achieved through a ubiquitous metabolic pathway called the methionine salvage pathway, which catalyzes conversion of the 5-methylthio-D-ribose moiety of S-methylthioadenosine to methionine (4 -7).In Klebsiela pneumoniae where all intermediates of the pathway have been identified (4 -7), metabolism of an aci-reductone is a branch point in the pathway ( Fig. 1) (8 -10). This molecule can undergo either a 1,2-oxygenlytic reaction to yield the ␣-keto acid precursor of methionine (Reaction 1) or a 1,3-oxygenlytic reaction to yield CO, formate, and methylthiopropionic acid (Reaction 2). The purpose of the off-pathway transformation of the aci-reductone (Reaction 2) is unclear. CO may simply be an easily cleared byproduct. Recent experiments in mammals, however, have established CO as a diffusible neurotransmitter, acting in a similar manner to that of nitric oxide (11,12). CO may also play a role as a messenger in bacteria.In a previous study we purified E-2, which catalyzes Reaction 2, to near homogeneity from K. pneumoniae (8). To study the structure and catalytic action of E-2, we decided to clone and overproduce the enzyme in Escherichia coli. To our surprise, E-2Ј which catalyzes Reaction 1, the other branch of the pathway, is overproduced in the same E. coli cells. In this report we describe the purification and characterization of both enzymes and demonstrate that their distinct catalytic and chromatographic properties result from binding different metals. EXPERIMENTAL PROCEDURESExpression and Purification of E-2 and E-2Ј-The E-2 gene was inserted at the initiator methionine codon of pET11a (13), a T7 RNA polymerase expression system. E. coli strain BL21, transformed with pET11a-E2, was grown in M9 medium (14) containing 100 mg/liter ampicillin at 37°C. When the absorbance at 600 nm reached 0.35, 0.3 mM isopropyl-D-thiogalactoside was added to induce E-2 expression at 25°C. The cells were incubated for an additional 12 h at 25°C, harvested by centrifugation, and stored frozen a...
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