The extents of formation of cobalt(II)-radical intermediates in the reactions with different substrates catalysed by the adenosylcobalamin-dependent enzyme ethanolamine ammonia-lyase.

1. The reactions of the adenosylcobalamin-dependent enzyme, ethanolamine ammonia-lyase, with the 'good' and 'relatively poor' substrates 2-aminoethanol and (S)-2-aminopropanol respectively, under conditions of saturation with substrate were investigated by rapid freezing in conjunction with electron paramagnetic resonance (e.p.r.) spectroscopy and by stopped-flow spectrophotometry. 2. In disagreement with earlier reports [Babior et al. (1972) J. Biol. Chem. 247, 4389-4392], it was found that the reaction of 2-aminoethanol gave an e.p.r. signal observed in rapid freezing experiments characteristic of a coupled Co(II)-free radical system. This signal was similar to, though not identical with, that obtained with (S)-2-aminopropanol. The steady-state level of the signal with 2-aminoethanol as substrate was 0.56 of that attained with (S)-2-aminopropanol. 3. The results of these e.p.r. experiments were shown to be consistent with stopped-flow data obtained under closely similar reaction conditions, the latter indicating a corresponding ratio of 0.64. The results also are consistent with those of a rapid wavelength scanning, stopped-flow spectrophotometric study [Hollaway et al. (1978) Eur. J. Biochem. 82, 143-154].

Chloroacetone as an active-site-directed inhibitor of the aliphatic amidase from Pseudomonas aeruginosa.

1. Chloroacetone (I) was shown to be an active-site-directed inhibitor of the aliphatic amidase (EC 3.5.1.4) from Pseudomonas aeruginosa strain PAC142.2. This inhibitor reacted with the enzyme in two stages: the first involving the reversible formation of an enzymically inactive species, EI, and the second the formation of a species, EX, from which enzymic activity could not be recovered. 3. Different types of kinetic experiment were conducted to test conformity of the reaction to the scheme: E + I k+1 Equilibrium k-1 EI Leads to K+2 EX A computer-based analysis of the results was carried out and values of the individual rate constants were determined. 4. No direct evidence for a binding step before the formation of EI could be obtained, as with [E]0 Less Than [I]0 the observed first-order rate constant for the formation of EI was directly proportional to the concentration of chloroacetone up to 1.2 mM (above this concentration the reaction became too rapid to follow even by the stopped-flow method developed to investigate fast inhibition). 5. The value of k+1 exhibited a bell-shaped pH-dependency with a maximum value of about 3 X 10(3) M-1. S-1 at pH6 and apparent pKa values of 7.8 and about 4.8.6. The values of k-1 and K+2 were similar and changed with the time of reaction from values of about 3 X 10(-3) S-1 (pH8.6) at short times to about one-sixth this value for longer periods of incubation. In this respect the simple reaction scheme is insufficient to describe the inhibition process. 7. The overall inhibition reaction is rapid, whether it is considered in relation to the expected chemical reactivity of chloroacetone, the rate of reaction of other enzymes with substrate analogues containing the chloromethyl group, or the rate of the amidase-catalysed hydrolysis of N-methylacetamide, a substrate that is nearly isosteric with chloroacetone. 8. Acetamide protected the amidase from inhibition by chloroacetone, and the concentration-dependence of the protection gave a value of an apparent dissociation constant similar to the Km value for this substrate. 9. Addition of acetamide to solutions of the species EI led to a slow recovery of activity. Recovery of active enzyme was also observed after dilution of a solution of EI in the absence of substrate. 10. The species EI is considered not to be a simple adsorption complex, and the possibilities are discussed that it may be a tetrahedral carbonyl adduct, a Schiff base (azomethine) or a complex in which the enzyme has undergone a structural change. The species EX is probably a derivative in which there is a covalent bond between a group in the enzyme and the C-1 atom of the inhibitor.

The number of functional active sites per molecule of the adenosylcobalamin-dependent enzyme, ethanolamine ammonia-lyase, as determined by a kinetic method.

1. A kinetic approach to the determination of the number of functional active sites per molecule of the adenosylcobalamin-dependent enzyme, ethanolamine ammonia-lyase, is described. 2. Time courses for formation and breakdown of a cob(II)alamin intermediate during reaction of the enzyme, fully saturated with adenosylcobalamin, with L-2-aminopropanol as substrate, were followed using a stopped-flow spectrophotometer under two conditions: (a) enzyme concentration much greater than that of substrate, (b) substrate concentration much greater than that of enzyme. 3. Results were analysed in terms of a three-step mechanism involving binding of substrate (k+1 step), cob(II)alamin formation (k+2 step) and cob(II)alamin breakdown (k+3 step). the kinetic scheme was shown to be sufficient to account for the observed time courses and rate constants of 80 s-1 (k+2) and 1.5 s-1 (k+3) were determined. 4. The number of active sites per enzyme molecule (n) was calculated from the kinetic data in three ways: (a) calculation from amplitude of absorbance measurement, (b) calculation from measurements of the values of rate constants and (c) analysis by computation of the kinetic data using the computer program FACSIMILE. A value for n close to 6 was calculated by each of these methods. This value is in disagreement with the literature value of about two sites per molecule but is consistent with the I6II6 subunit structure of the enzyme. 5. Kinetic analysis of data from experiments in which the adenosylcobalamin concentration was varied while substrate and enzyme concentrations remained constant showed that all the active sites function with identical rate constants. 6. The principle and mathematical basis of the kinetic method for determining the value of n is given as an Appendix.

The subunit structure of rabbit skeletal-muscle phosphofructokinase and the amino acid sequence of the tryptic peptide containing the highly reactive thiol group.

1. The single highly reactive (class I) thiol group per 80000-mol.wt. subunit of skeletal-muscle phosphofructokinase was specifically carboxymethylated with iodo[2-14C]acetate, and after denaturation the remaining thiol groups were carboxymethylated with bromo[2-3H]acetate. After tryptic digestion and peptide 'mapping' it was found that the 14C radioactivity was in a spot that did not contain significant amounts of 3H radioactivity, so it is concluded that there is not a second, 'buried' cysteine residue within a sequence identical with that of the class-I cysteine peptide. 2. The total number of tryptic peptides as well as the number of those containing cysteine, histidine or tryptophan were inconsistent with the smallest polypeptide chain of phosphofructokinase (mol.wt. about 80000) being composed of two identical amino acid sequences. 3. The amino acid sequence of the tryptic peptide containing the class-I thiol group was shown to be Cys-Lys-Asp-Phe-Arg. This sequence is compared with part of the sequence containing the highly reactive thiol group of phosphorylase.

The reduction by dithionite of Fe(III) myoglobin derivatives with different ligands attached to the iron atom. A study by rapid-wavelength-scanning stopped-flow spectrophotometry.

1. The reductions of a number of sperm whale Fe(III) myoglobin-ligand complexes by sodium dithionite in a phosphate buffer pH 6.4, were investigated by using rapid-wavelength-scanning stopped-flow spectrophotometry. The ligands were azide, cyanide, fluoride, imidazole, thiocyanate and water. 2. The reduction of Fe(III) myoglobin cyanide led to the transient formation of Fe(II) myoglobin cyanide but no intermediate species were observable during the reductions of the other derivatives. The final product of the reaction in all cases was unliganded Fe(II)myoglobin. 3. Invesigation of the effect of dithionite concentration on the rate of reduction indicated that the SO2- radical ion was the active species in reducing the azide, cyanide, fluoride and thiocyanate derivatives. 4. Comparison of the observed rates of reduction at different ligand concentrations with those predicted for a pathway of reduction involving prior dissociation of the ligand, allowed us to estimate the rate of reduction with the ligand in position (outer-sphere reduction). There was a large variation in the relative rates of outer-sphere reduction in the order imidazole greater than CN- greater than SCN- greater than N3- greater than F-. The fluoride derivative was so resistant to outer-sphere reduction that the reaction with SO2- proceeded only by a pathway involving dissociation of F- before reduction. It was calculated that any direct reduction of this complex was at least 100 times slower than that of the azide derivative. 5. The results are discussed in terms of the possible rôle of the axial ligands in haem proteins and it is suggested that the pathway of the electron to the Fe(III) centre may be via the ppi orbitals of these ligands.

An investigation of the nicotinamide-adenine dinucleotide-induced 'tightening' of the structure of glyceraldehyde 3-phosphate dehydrogenase.

An investigation was made of the effect of NAD+ analogues on subunit interactions in yeast and rabbit muscle glyceraldehyde 3-phosphate dehydrogenases by using the subunit exchange (hybridization) method described previously [e.g. see Osborne & Hollaway (1975) Biochem. J. 151, 37-45]. The ligands ATP, ITP, ADP, AMP, cyclic AMP and ADP-ribose like NADH, all caused an apparent weakening of intramolecular subunit interactions, whereas NAD+ caused an apparent increase in the stability of the tetrameric enzyme molecules. A mixture of NMN and AMP, although it did not simulate completely the NAD+-induced 'tightening' of the enzyme structure, did result in a more than 20-fold decrease in the rate of subunit exchange compared with that in the presence of AMP alone. These results show that occupancy of the NMN subsite of the enzyme NAD+-binding site is insufficient in itself to give the marked tightening of the enzyme structure induced by NAD+. The 'tightening' effect is specific in that it seems to require a phosphodiester link between NMN and ADP-ribose. These effects are discussed in terms of the detailed X-ray structure of the lobster holoenzyme [Buehner et al. (1974) J. Mol. Biol. 90, 25-49].

The investigation of substrate-induced changes in subunit interactions in glyceraldehyde 3-phosphate dehydrogenases by measurement of the kinetics and thermodynamics of subunit exchange.

An investigation was made of changes in subunit interactions in glyceraldehyde 3-phosphate dehydrogenase on binding NAD+, NADH and other substrates by using the previously developed method of measurement of rates and extent of subunit exchange between the rabbit enzyme (R4), yeast enzyme (Y4) and rabbit-yeast hybrid (R2Y2) [Osborne & Hollaway (1974) Biochem. J. 143, 651-662]. The free energy of activation for the conversion of tetramer into dimer for the rabbit enzyme (R4 leads to 2R2) is increased by at least 12kJ/mol in the presence of NAD+. This increase is interpreted in terms of an NAD+-induced 'tightening' of the tetrameric structure probably involving increased interaction at the subunit interfaces across the QR plane of the molecule [see Buehner et al. (1974) J. Mol. Biol. 82, 563-585]. This tightening of the structure only occurs on binding the third NAD+ molecule to a given enzyme molecule. Conversely, binding of NADH causes a decrease in the free energy of activation for the R4 leads to 2R2 and Y4 leads to 2Y2 conversions by at least 10kJ/mol. This is interpreted as a NADH-induced 'loosening' of the structures arising from decreased interactions across the subunit interfaces involving the QR dissociation plane. In the presence of NADH the increase in the rate of subunit exchange is such that it is not possible to separate the hybrid from the other species if electrophoresis is carried out with NADH in the separation media. In the presence of a mixture of NADH and NAD+ the effect of NAD+ on subunit exchange is dominant. The results are discussed in terms of the known co-operativty between binding sites in glyceraldehyde 3-phosphate dehydrogenases.

The hybridization of glyceraldehyde 3-phosphate dehydrogenases from rabbit muscle and yeast. Kinetics and thermodynamics of the reaction and isolation of the hybrid.

A kinetic and thermodynamic study was made of the formation of the hybrid (R(2)Y(2)) glyceraldehyde 3-phosphate dehydrogenase from the yeast (Y(4)) and rabbit (R(4)) enzymes. The values of the thermodynamic parameters for the equilibrium between R(4), Y(4) and R(2)Y(2) suggest that the R(2)-R(2) and Y(2)-Y(2) interactions are similar. However, the failure to observe the RY(3) and R(3)Y hybrids is interpreted in terms of differences at the interfaces of the R-R and Y-Y interactions (the glyceraldehyde 3-phosphate dehydrogenase molecule being regarded as a dimer of dimers). The kinetics of formation of the R(2)Y(2) hybrid were studied and a model was proposed to account for the results. Best-fit values for the rate constants of the individual steps were evaluated by computer simulation, and the rate-limiting steps were identified as the dissociation of tetramers to dimers. It is proposed that the cleavage plane for dissociation of the tetramers corresponds to the region of low electron density through the centre of the molecule in the X-ray-crystallographic structure for human glyceraldehyde 3-phosphate dehydrogenase (Watson et al., 1972), which is probably the plane containing the Q and R axes in the lobster enzyme (Buehner et al., 1974). The R(2)Y(2) hybrid was isolated in milligram amounts by ion-exchange chromatography and its rate of reversion to the native enzyme was shown to be consistent with the kinetic model proposed from the hybrid-formation experiments.