The use of DL-[2-2H]lactate in steady-state measurements of ferricyanide reduction by flavocytochrome b2 at 30 'C has previously yielded an isotope effect of 5 [F. Lederer (1974) Eur. J . Biochem. 46, 393-3991. We report here studies carried out at 5°C with 1~-[2-~H]lactate, where flavin and heme reduction were observed in the stopped-flow apparatus, in the absence of acceptor. The generally biphasic reduction curves were analysed according to a new mathematical treatment which allowed us to derive microscopic constants from initial reduction rates. It has thus been possible to determine an isotope effect of 8 on flavin reduction, 6 on heme reduction, compared to 4 in the steady state. Consequently, two slightly rate-limiting steps occur after the first one where the a-hydrogen is abstracted. It has also been possible to calculate the substrate association and dissociation rate constants for intact enzyme.The studies were carried out in parallel on intact and cleaved cytochrome b2. The results suggest that proteolysis affects essentially the steps involved in flavin reduction, and not intramolecular electron transfer steps. Moreover, the experimental data obtained at low rates of electron entry have led us to reexamine a previously proposed scheme for electron transfer [Capeillere-Blandin, Bray, Iwatsubo and Labeyrie (1975) Eur. J . Biochem. 54, 549-5661, An alternative model based on computer-simulation studies will be presented in a paper in this journal. . This result suggested that the rate-determining step was more precisely the abstraction of the a-hydrogen, before electron transfer to flavin. Since, however, the magnitude of an isotope effect depends on the extent of proton transfer in the transition state [3], a value of 5 did not preclude that some other step was partially rate-limiting; consequently it appeared of interest to carry out a study of prosthetic group reduction by deuterolac-* Deceased.Enzyme. L-Lactate : cytochrome c oxidoreductase (EC 1 .I .2.3).tate, using rapid kinetic methods. This study is the subject of the present paper. Design of the experiments and their interpretation were facilitated by a recent investigation which led the authors to propose a detailed kinetic scheme for the series of electron transfers leading from oxidized enzyme and lactate to fully reduced enzyme and pyruvate [4,5]. While that investigation provided guidelines for the present work, the use of [2-2H]lactate conversely appeared to offer a possible means of verifying the scheme since the replacement of hydrogen by deuterium would selectively modify the rate of certain steps.The present study was carried out with L -[~-~H ] -lactate, instead of the racemic lactate which had been used previously [2]. Moreover, the behaviour of both forms of flavocytochrome 62 was examined. The intact enzyme and the cleaved one differ by a number of steady-state kinetic parameters [6,7]. In particular, the intact enzyme specific activity is 2.5-fold higher than that of the cleaved one [7], the substrate K, is about 3-fold smaller ...

L-Glutamine:D-fructose-6-phosphate amidotransferase (glucosamine synthetase) has been purified to homogeneity from Escherichia coli. A subunit molecular weight of 70,800 was estimated by gel electrophoresis in sodium dodecyl sulfate. Pure glucosamine synthetase did not exhibit detectable NH3-dependent activity and did not catalyze the reverse reaction, as reported for more impure preparations [Gosh, S., Blumenthal, H. J., Davidson, E., & Roseman, S. (1960) J. Biol. Chem. 235, 1265]. The enzyme has a Km of 2 mM for fructose 6-phosphate, a Km of 0.4 mM for glutamine, and a turnover number of 1140 min-1. The amino-terminal sequence confirmed the identification of residues 2-26 of the translated E. coli glmS sequence [Walker, J. E., Gay, J., Saraste, M., & Eberle, N. (1984) Biochem. J. 224, 799]. Methionine-1 is therefore removed by processing in vivo, leaving cysteine as the NH2-terminal residue. The enzyme was inactivated by the glutamine analogue 6-diazo-5-oxo-L-norleucine (DON) and by iodoacetamide. Glucosamine synthetase exhibited half-of-the-sites reactivity when incubated with DON in the absence of fructose 6-phosphate. In its presence, inactivation with [6-14C]DON was accompanied by incorporation of 1 equiv of inhibitor per enzyme subunit. From this behavior, a dimeric structure was tentatively assigned to the native enzyme. The site of reaction with DON was the NH2-terminal cysteine residue as shown by Edman degradation.

The membrane-bound acetylcholine receptor from Torpedo marmorata was photolabeled by the noncompetitive channel blocker [3H]chlorpromazine under equilibrium conditions in the presence of the agonist carbamoylcholine. The amount of radioactivity incorporated into all subunits was reduced by addition of phencyclidine, a specific ligand for the high-affinity site for noncompetitive blockers. The labeled p chain was purified and digested with trypsin or CNBr, and the resulting fragments were fractionated by high-performance liquid chromatography. Sequence analysis resulted in the identification of Ser-254 and Leu-257 as residues labeled by [3H]chlorpr~mazine in a phencyclidine-sensitive manner. These residues are located in the hydrophobic and potentially transmembrane segment M I1 of the chain, a region homologous to that containing the chlorpromazine-labeled Ser-262 in the 6 chain [Giraudat, J., Dennis, M.These results show that homologous regions of different receptor subunits contribute to the unique high-affinity site for noncompetitive blockers, a finding consistent with the location of this site on the axis of symmetry of the receptor molecule.x e nicotinic acetylcholine receptor (AcChR)' from fish electric organ and vertebrate neuromuscular junction is a ABSTRACT: We have previously described a specific protease in turkey erythrocytes that converts the larger 50-kDa (P50) form of the P,-adrenoceptor to a smaller 40-kDa (P40) form [Jurss, R., Hekman, M., &

The role of Tyr-143 in the catalytic cycle of flavocytochrome b2 (L-lactate:cytochrome c oxidoreductase) has been examined by replacement of this residue with phenylalanine. The electron-transfer steps in wild-type and mutant flavocytochromes b2 have been investigated by using steady-state and stopped-flow kinetic methods. The most significant effect of the Tyr-143----Phe mutation is a change in the rate-determining step in the reduction of the enzyme. For wild-type enzyme the main rate-determining step is proton abstraction at the C-2 position of lactate, as shown by the 2H kinetic-isotope effect. However, for the mutant enzyme it is clear that the slowest step is interdomain electron transfer between the FMN and haem prosthetic groups. In fact, the rate of haem reduction by lactate, as determined by the stopped-flow method, is decreased by more than 20-fold, from 445 +/- 50 s-1 (25 degrees C, pH 7.5) in the wild-type enzyme to 21 +/- 2 s-1 in the mutant enzyme. Decreases in kinetic-isotope effects seen with [2-2H]lactate for mutant enzyme compared with wild-type, both for flavin reduction (from 8.1 +/- 1.4 to 4.3 +/- 0.8) and for haem reduction (from 6.3 +/- 1.2 to 1.6 +/- 0.5) also provide support for a change in the nature of the rate-determining step. Other kinetic parameters determined by stopped-flow methods and with two external electron acceptors (cytochrome c and ferricyanide) under steady-state conditions are all consistent with this mutation having a dramatic effect on interdomain electron transfer. We conclude that Tyr-143, an active-site residue which lies between the flavodehydrogenase and cytochrome domains of flavocytochrome b2, plays a key role in facilitating electron transfer between FMN and haem groups.