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J Knappe

Publications and source records attributed to J Knappe.

At least 19 recordsLinked to original sources
PubMed1994

Adenosylmethionine-dependent synthesis of the glycyl radical in pyruvate formate-lyase by abstraction of the glycine C-2 pro-S hydrogen atom. Studies of [2H]glycine-substituted enzyme and peptides homologous to the glycine 734 site.

The active form of pyruvate formate-lyase (PFL) from Escherichia coli contains a glycyl radical in position 734 of the polypeptide chain which is produced post-translationally by pyruvate formate-lyase-activating enzyme (PFL activase) using S-adenosylmethionine (AdoMet) and dihydroflavodoxin as co-substrates (Wagner, A.F. V., Frey, M., Neugebauer, F.A., Schäfer, W., and Knappe, J. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 996-1000). Studying radical synthesis with [2-2H]glycine-labeled PFL, we have now found stoichiometric incorporation of a 2H atom into the 5'-deoxyadenosine (dAdo) co-product via mass and NMR spectroscopic analyses. Furthermore, a series of peptides homologous to the Gly-734 site of PFL have been synthesized for analyzing recognition determinants of PFL activase. Peptides that proved active as substrates (monitored by [14C]dAdo formation from [14C]AdoMet) were also competitive inhibitors of PFL conversion to the radical form. In the sequence of the standard peptide Arg-Val-Ser-Gly-Tyr-Ala-Val, which corresponds to amino acid residues 731-737 of PFL, the Gly residue was replaceable by D-Ala (actually displaying enhanced efficiency), whereas a normal Ala totally abolished the interaction with PFL activase. Our results show that the radical in pyruvate formatelyase is produced by stereospecific abstraction of the pro-S hydrogen of glycine 734 by the 5'-dAdo radical generated in the active center of PFL activase. Gly-734 is probably located in a beta-turn segment of the protein.

Acetyltransferases↗
PubMed1992

Ultrastructure and pyruvate formate-lyase radical quenching property of the multienzymic AdhE protein of Escherichia coli.

The AdhE protein of Escherichia coli is a homopolymer of 96-kDa subunits harboring three Fe(2+)-dependent catalytic functions: acetaldehyde-CoA dehydrogenase, alcohol dehydrogenase, and pyruvate formatelyase (PFL) deactivase. By negative staining electron microscopy, we determined a helical assembly of 20-60 subunits into rods of 45-120 nm in length. The subunit packing is widened along the helix axis when Fe2+ and NAD are present. Chymotrypsin dissects the AdhE polypeptide between Phe762 and Ser763, thereby retaining the alcohol dehydrogenase activity on the NH2-terminal core, but destroying all other activities. PFL deactivation, i.e. quenching of the glycyl radical in PFL by the AdhE protein, was examined with respect to cofactor involvements (Fe2+, NAD, and CoA). This process is coupled to NAD reduction and requires the intact CoA sulfhydryl group. Pyruvate and NADH are inhibitors that affect the steady-state level of the radical form of PFL in a reconstituted interconversion cycle. Studies of cell cultures found that PFL deactivation in situ is initiated at redox potentials of greater than or equal to +100 mV. Our results provide insights into the structure/function organization of the AdhE multienzyme and give a rationale for how its PFL radical quenching activity may be suppressed in situ to enable effective glucose fermentation.

Acetyltransferases↗
PubMed1992

The free radical in pyruvate formate-lyase is located on glycine-734.

Pyruvate formate-lyase (acetyl-CoA:formate C-acetyltransferase, EC 2.3.1.54) from anaerobic Escherichia coli cells converts pyruvate to acetyl-CoA and formate by a unique homolytic mechanism that involves a free radical harbored in the protein structure. By EPR spectroscopy of selectively 13C-labeled enzyme, the radical (g = 2.0037) has been assigned to carbon-2 of a glycine residue. Estimated hyperfine coupling constants to the central 13C nucleus (A parallel = 4.9 mT and A perpendicular = 0.1 mT) and to 13C nuclei in alpha and beta positions agree with literature data for glycine radical models. N-coupling was verified through uniform 15N-labeling. The large 1H hyperfine splitting (1.5 mT) dominating the EPR spectrum was assigned to the alpha proton, which in the enzyme radical is readily solvent-exchangeable. Oxygen destruction of the radical produced two unique fragments (82 and 3 kDa) of the constituent polypeptide chain. The N-terminal block on the small fragment was identified by mass spectrometry as an oxalyl residue that derives from Gly-734, thus assigning the primary structural glycyl radical position. The carbon-centered radical is probably resonance-stabilized through the adjacent carboxamide groups in the polypeptide main chain and could be comparable energetically with other known protein radicals carrying the unpaired electron in tyrosine or tryptophan residues.

Acetyltransferases↗
PubMed1991

Pyruvate-formate-lyase-deactivase and acetyl-CoA reductase activities of Escherichia coli reside on a polymeric protein particle encoded by adhE.

A 4.8 kb DNA-fragment was cloned and sequenced encompassing the structural gene of PFL-deactivase (2.7 kb) and 2 kb of the 5' flanking region that contains the elements for anaerobic induction. A mutant lacking deactivase was shown to require exogenous electron acceptors for anaerobic growth with glucose. This revealed the identity of PFL-deactivase with the alcohol and acetaldehyde dehydrogenases of E. coli. The multienzyme represents a homopolymeric protein (approximately 40 x 96 kDa) requiring Fe2+ for all functions.

Alcohol Dehydrogenase↗
PubMed1990

[The scientific basis and evaluation of the present-day concept of risk factors and their significance for the prevention of chronic non-communicable diseases].

Chronic diseases increased in their significance, to them first of all belong cardiovascular diseases. They particularly restrict a further increase of the life-expectancy of the persons older than 40 years. In the judgement of the development of these groups of diseases during the last decades the so-called concept of risk factors was developed, which obtained particular significance for the myocardial infarction and the chronic cardiovascular diseases. Though the epidemiologic investigations up to now could cover only correlations and not causal connections, the latter - that means causal conditions - seem to get an increasing support by results of the adequate basic research. Still more impressionable is, however, the regression of the mortality due to cardiovascular diseases which took place during recent years in connection with the changes of the living habits in several countries of the earth. From the results of our own research the particular urgencies concerning the preventive medicine in our country are described which must consist in changes of the living habits, in particular of the habits of nutrition and the abuse of coffee, tea, tobacco, alcohol, etc., moreover also in personal behaviour. Independent of this genetic factors certainly play a decisive part. Apart from the individual strategy mass-strategic aspects will obtain a greater importance in the following years.

Aged↗
PubMed1990

A radical-chemical route to acetyl-CoA: the anaerobically induced pyruvate formate-lyase system of Escherichia coli.

Anaerobically growing Escherichia coli cells contain the enzyme pyruvate formate-lyase which catalyses the non-oxidative cleavage of pyruvate to acetyl-CoA and formate. The enzyme is subject to interconversion between inactive and active forms. The active form contains an oxygen-sensitive organic free radical located on the polypeptide chain which is essential for catalysis. It affords a novel homolytic C-C bond cleavage of the pyruvate substrate. The radical is generated by an iron-dependent converter enzyme which requires reduced flavodoxin and adenosyl methionine as co-substrates and pyruvate as a positive allosteric effector. A second converter enzyme, also iron-dependent, accomplishes the removal of the radical. This post-translational interconversion cycle controls the activity state of pyruvate formate-lyase in the anaerobic cell. Anaerobic conditions also regulate pyruvate formate-lyase at the level of gene expression. Multiple promoters are responsible for effecting a twelve to fifteen fold induction and they are coordinately controlled in response to the oxygen and metabolic status of the cell by sequences which are located far upstream of the pfl coding region. The transcription factor Fnr has been identified as being responsible for part of the anaerobic control of pfl expression, probably through direct interaction with the upstream sequences. In contrast, the expression of the gene encoding the first iron-dependent converter enzyme is unaffected by anaerobiosis and is independent of the Fnr protein.

Acetyl Coenzyme A↗
PubMed1989

The free radical of pyruvate formate-lyase. Characterization by EPR spectroscopy and involvement in catalysis as studied with the substrate-analogue hypophosphite.

The first-derivative EPR spectrum of the active form of Escherichia coli pyruvate formate-lyase shows an asymmetric doublet with partially resolved hyperfine splittings (g = 2.0037). Isotope substitution studies demonstrated couplings of a carbon-centered unpaired electron to a solvent-exchangeable proton (a = 1.5 mT) and to further hydrogen nuclei (a = 0.36 and 0.57 mT). By selective incorporation of unlabelled tyrosine into 2H-labelled enzyme protein, a tyrosyl radical structure has been ruled out. Circumstantial evidence indicates that the organic free radical, which also displays an ultraviolet absorption signal at 365 nm, is located on a standard amino acid residue of the polypeptide chain. EPR signal quantification found a stoichiometry of 1 spin per active site. The formate analogue hypophosphite has been characterized as a specific kcat inhibitor of pyruvate formate-lyase which destroys the enzyme radical. Protein-linked 1-hydroxyethylphosphonate was previously described as the dead-end product after reaction of the analogue with the intermediary acetyl-enzyme form of the catalytic cycle [W. Plaga et al. (1988) Eur. J. Biochem. 178, 445-450]. EPR spectroscopy of this system has now identified the corresponding alpha-phosphoryl radical as a reaction intermediate [g = 2.0032; a(P) = 2.72 mT, a(3H) = 1.96 mT]; it showed a half-life of about 20 min at 0 degrees C. This finding proves that the enzyme radical is a hydrogen-atom-transferring coenzymic element.

Acetyltransferases↗
PubMed1989

[Dose-reduced antihypertensive agents--use in complex nonmedicamentous therapy of hypertension].

In order to estimate the influence of a non-medicamentous therapy (CNT) on the consumption of medicaments and coronary risk in high blood pressure 73 hypertensives of a medicamentously stabilized CNT-group were examined in comparison to a group of the same size of patients with hypertension who were managed exclusively medicamentously for behaviour of blood pressure, need of antihypertensive drugs and changes of hypertension-associated risk factors. After an exactly controlled 6-month treatment hypertensives with additionally recommended far-reaching CNT showed an economization of medicaments by scarcely the half in comparison to the reference group. By means of suitable control methods a causal non-medicamentously conditioned decrease of blood pressure could be excluded. A different need of antihypertensive drugs was simulated by the exacter intake of medicaments in the index-patients. Notwithstanding the metabolic effects of the additional therapy have induced a positive change of atherogenic lipids. The examinations indicate in general the difficulty of the judgement of efficacy of non-medicamentous therapeutic measures in connection with a rational dose-reduced long-term therapy with antihypertensive drugs.

Adult↗
PubMed1988

Catalytic-site mapping of pyruvate formate lyase. Hypophosphite reaction on the acetyl-enzyme intermediate affords carbon-phosphorus bond synthesis (1-hydroxyethylphosphonate).

Pyruvate formate-lyase of Escherichia coli cells, a homodimeric protein of 2 x 85 kDa, is distinguished by the property of containing a stable organic free radical (g = 2.0037) in its resting state. The enzyme (E-SH) achieves pyruvate conversion to acetyl-CoA via two distinct half-reactions (E-SH + pyruvate in equilibrium E-S-acetyl + formate; E-S-acetyl + CoA in equilibrium E-SH + acetyl-CoA), the first of which has been proposed to involve reversible homolytic carbon-carbon bond cleavage [J. Knappe et al. (1984) Proc. Natl Acad. Sci. USA 81, 1332-1335]. Present studies identified Cys-419 as the covalent-catalytic cysteinyl residue via CNBr fragmentation of E-S-[14C]acetyl and radio-sequencing of the isolated peptide CB-Ac (amino acid residues 406-423). Reaction of the formate analogue hypophosphite with E-S-acetyl was investigated and found to produce 1-hydroxyethylphosphonate with a thioester linkage to the adjacent Cys-418. The structure was determined from the chymotryptic peptide CH-P (amino acid residues 415-425), using 31P-NMR spectroscopy (delta = 44 ppm) and by chemical characterisation through degradation into 1-hydroxyethylphosphonate with phosphodiesterase or bromine. This novel P-C-bond synthesis involves the enzyme-based free radical and is proposed to resemble the physiological C-C-bond synthesis (pyruvate production) from formate and E-S-acetyl. These findings are interpreted as proof of a radical mechanism for the action of pyruvate formate-lyase. The central Cys-418/Cys-419 pair of the active site shows a distinctive thiolate property even in the inactive (nonradical) form of the enzyme, as determined using an iodoacetate probe.

Acetyl Coenzyme A↗
PubMed1988

[Nifedipine therapy in uncomplicated essential hypertension and hypertensive problem cases].

In altogether 54 patients with high blood pressure of the clinical degrees of severity I-IV the blood pressure-reducing effectiveness of the calcium antagonist nifedipine was investigated both in the framework of an oral long-term therapy and especially in problematic situations of high blood pressure. According to the results of own treatment nifedipine is particularly suited for the antihypertensive combination therapy with beta-receptor blockers. Monotherapeutically favourable possibilities of application exist in hypokinetic types of the regulation of circulation as well as in elderly hypertensives. Therapy-resistant as well as crisis-like developments of high blood pressure react with excessive, well tolerable and persisting reductions of blood pressure. Above all with regard to this still little considered indication in the meantime nifedipine represents an unavoidable enrichment in the therapeutic basis spectre of the hypertonus.

Adult↗
PubMed1988

[Status and perspectives of prevention in coronary heart disease].

The outstanding objective prerequisites for the protection of life and health of the whole population, which are at our disposal, have quite significantly widened the possibilities for individual decisions and actions concerning the influence on health and functional capacity as well as responsibility and cooperation of every citizen. Comprehensively, also in our country the readiness of all citizens must be stimulated to form the personal way of life according to the scientific knowledge fully using the abundantly given possibilities. In this respect we too have to solve still great problems.

Coronary Disease↗
PubMed1988

Primary structures of Escherichia coli pyruvate formate-lyase and pyruvate-formate-lyase-activating enzyme deduced from the DNA nucleotide sequences.

The structural gene of pyruvate formate-lyase (pfl) and that of pyruvate-formate-lyase-activating enzyme were shown to be adjacent on the chromosomal map of Escherichia coli. DNA sequencing was performed along a stretch of 3592 nucleotides to obtain the amino acid sequences of both proteins. The derived primary structures (759 and 245 residues) were confirmed by partial structure analyses on the purified proteins. The open reading frames are separated by a 194-nucleotide stretch, and their flanking regions include signal elements that are compatible with separate control of protein synthesis from the two genes.

Acetyltransferases↗
PubMed1988

[Results of a 2-year intervention for the modification of atherosclerosis precursors in school children].

801 children aged 10-11 years were examined in 1984, before an allocation in two groups (intervention vs. control). The scope of the intervention was to modify mean values of blood pressure, total cholesterol and smoking through relevant modification of knowledge and behavior. An intermediate assessment, conducted 2 years after the first exam, showed better knowledge regarding cardiovascular diseases and more sport practice in the intervention group, although smoking habits were only slightly modified. Diet, body weight, blood pressure and blood lipid levels did not show any significative differences. The study is now ongoing.

Anthropometry↗
PubMed1987

[Prevention of ischemic heart disease--experiences and conclusions from the Erfurt intervention study].

The concept of the primary prevention of the ischaemic heart disease by modification of the main factors of risk smoking of cigarettes, hypercholesterolaemia and high blood pressure at present got an up to now not achieved degree of probability. Nowadays we no more doubt about the fact that profound changes of nutrition, on optimum treatment of hypertension and a regression of the consumption of cigarettes might have a clear influence on case rates and mortality rates of ischaemic heart disease, but also of other chronic diseases. Also for our country the principal possibility of a preventive influence was confirmed (Erfurt intervention study). The prevention of the ischaemic heart disease by modification of the main factors of risk as an integrated constituent of medical activity by far more than up to now must be introduced into the principal medical care. But in the first place prevention is also a task of the state and the whole society.

Coronary Disease↗
PubMed1987

[Clinical and paraclinical findings on status following prosthetic heart valve replacement with special reference to the behavior of pulmonary pressure].

Retrospectively were evaluated clinical findings of 151 patients with condition after replacement of artificial cardiac valves (Björk-Shiley), who were treated in our department of cardiology in 1986. The patients with mitral valve replacement (MVR) and mitral valve and aortic valve replacement showed the highest rate of individualisation in comparison to aortic valve replacement. 18 patients showed complications and acute deteriorations, respectively, two of which had a fatal course. In 53 patients the bicycle-ergometric exercises were performed with measurement of the pulmonary pressure. Of these patients the majority indeed subjectively improved in their behaviour, however, the objective exercise tolerance was altogether low and the rate of invalidisation increased in comparison to the preoperative rate. The pulmonary pressure at rest in the majority of the patients was reduced mostly in dependence on the initial values. Increases appeared only sporadically. Under exercise, however, particularly the patients with mitral valve replacement showed pathological increases of pressure. Altogether the best results showed patients with aortic valve replacement also with regard to the shrinking of the radiologically established size of the heart.

Adolescent↗