The first-derivative EPR spectrum of the active form of Escherichiu 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 I spin per active site.The formate analogue hypophosphite has been characterized as a specific k,,, inhibitor of pyruvate formatelyase 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,. EPR spectroscopy of this system has now identified the corresponding a-phosphoryl radical as a reaction intermediate [g = 2.0032; u(P) = 2.72 mT, a(3H) = 1.96 mT]; it showed a half-life of about 20 min at 0°C. This finding proves that the enzyme radical is a hydrogen-atomtransferring coenzymic element.Pyruvate formate-lyase is the key enzyme of the anaerobic glucose degradation route in Escherichia coli cells [l]. It shows unique mechanisms of enzyme regulation and catalysis. Transcription of the structural gene (~f l )is highly controlled by anaerobic induction [2]. At the post-translational level, pyruvate formate-lyase is interconverted by specific converter enzymes between a fully inactive form (Ei) and an active form (E,) that contains a persistent organic free radical [3]. Pyruvate is nonoxidatively processed to acetyl-CoA by a two-step reaction pattern with an intermediary acetyl-enzyme:(2) Addressing structure/function relationships, we have recently mapped the covalent catalytic SH-group as the Cys-419 residue of the polypeptide chain [4]. These studies also showed that the acetyl-enzyme intermediate will react with the formate analogue hypophosphite to produce l-hydroxyethylphosphonate, linked as a thioester to Cys-418. The C-P bond-forming process, which resembles pyruvate synthesis from formate (reversal of Eqn l), has now been investigated by EPR spectroscopy. We have identified a substrate-based radical intermediate, thus demonstrating for the first time a functional role of the free radical of pyruvate formate-lyase. Our findings substantiate the notion that the reversible C-C bond cleavage/synthesis in the first half-reaction of the catalytic cycle occurs by a radical mechanism.We also report in this paper the EPR and ultraviolet spectroscopic characterization of the enzyme radical, its quantification, and several isotope substitution experiments for approachi...

The new CoaguChek XS system is designed for use in patient selftesting. It is the successor of the current CoaguChek S system. The detection principle is based on the amperometric measurement of the thrombin activity initiated by starting the coagulation cascade using a human recombinant thromboplastin. This study was performed to assign the International SEnsitivity Index (ISI) to the new test according to the WHO guidelines for thromboplastins and plasmas used to control anticoagulant therapy, and to establish the measuring range of the new system. At four study sites a total of 90 samples of normal donors and 291 samples of warfarin-, phenprocoumon- or acenocoumarol-treated patients were included in the study. The ISI value of the new test was assigned against the human recombinant reference thromboplastin rTF/95 at each site using the samples from stabilized patients in the International Normalized Ratio (INR) range between 1.5 and 4.5 only. The new point-of-care system's measuring range between 0.8 and 8 INR was calibrated against the mean INR of rTF/95 and AD149 using polynomial regression. ISIs were (CV of the slope): Site 1: ISI 0.99 (1.1%); Site 2: ISI 1.02 (2.0%); Site 3: ISI 1.03 (1.1%); Site 4: ISI 1.00 (1.4%). All regression lines calculated from patient-only data pass through the normal donor data points. All CVs of the slopes of the orthogonal regression lines are well below 3%, thus fulfilling the requirements of the WHO guidelines. The mean ISI for the new CoaguChek XS PT Test is 1.01.

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