1. The five glycolyticenzymes glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase and pyruvate kinase were each purified from extracts of Zymomonas mobilis cells, by using dye-ligand chromatography as the principal step. Two procedures, producing three and two of the enzymes respectively, are described in detail. 2. Z. mobilis glyceraldehyde-phosphate dehydrogenase was found to be similar in most respects to the enzyme from other sources, except for having a slightly larger subunit size. 3. Phosphoglycerate kinase has properties typical for this enzyme; however, it did not show the sulphate activation effects characteristic of this enzyme from most other sources. 4. Phosphoglycerate mutase is a dimer, partially independent of 2,3-bisphosphoglycerate, and has a high specific activity. 5. Enolase was found to be octameric; otherwise its properties were very similar to those of the yeast enzyme. 6. Pyruvate kinase is unusual in being dimeric, and not requiring K+ for activity. It is not allosterically activated by sugar phosphates, having a high activity in the absence of any effectors. 7. Some quantitative differences in the relative amounts of these enzymes, compared with eukaryotic species, are ascribed to the fact that Z. mobilis utilizes the Entner-Doudoroff pathway rather than the morecommon Embden-Meyerhoff glycolytic route.

We have detected a flavonoid 3’,5‘-hydroxylase (F3’,5’H) in the microsomal fraction of Petunia hybrida flowers. Activity varied with the development of flowers, peaking immediately prior to and during anthesis, but was absent in mature flowers. F3’,5’H activity in flower extracts from genetically defined floral color mutants correlated strictly with the genotypes H f l and H f 2 . No activity was detected in flowers from mutants homozygous recessive for both alleles. F3’,5’H activity was dependent on NADPH and molecular oxygen; there was only slight activity with NADH. The enzyme catalyzes the hydroxylation of 5,7,4‘-trihydroxyflavonone at the 3’ and 5‘ positions, and of 5,7,3‘,4‘-tetrahydroxyflavonone and dihydroquercetin at the 5’ position. Hydroxylase activity was inhibited by plant growth regulators (1-aminobenzotriazole and tetcyclacis) and by CO, N-ethylmaleimide, diethyldithiocarbamate, and cytochrome (Cyt) c. Activity was not affected by diethylpyrocarbonate or phenylmethylsulfonyl fluoride, but was enhanced by 2-mercaptoethanol. A polyclonal antibody that inhibits higher plant NADPHCyt P450 reductase inhibited the F3’,5’H. The data are consistent with the suggestion that the P. hybrida F3’,5’H i s a monooxygenase consisting of a Cyt P450 and a NADPH-Cyt P-450 reductase. Cyts P450 were detected in microsomal membranes and in solubilized detergent extracts of these membranes. F3’,5’H activity was sensitive to low concentrations of all detergents tested, and therefore solubilization of the active enzyme was not achieved. Reaction products other than flavanones were observed in F3’,5‘H assays and these may be formed by enzymic oxidation of flavanones. The possibility of a microsomal flavone synthase of a type that has not been described in P. hybrida i s discussed.

Eduard Buchner’s discovery of cell-free metabolism 100 years ago was a landmark in biochemistry; it can be said to be the start of detailed metabolic studies. The fact that a soluble fraction of yeast, which he called “zymase”, was able to produce ethanol from glucose was revolutionary, and finally put paid to the idea that living cells were essential for fermentation (Buchner, 1897). Later, this proteincontaining extract was recognized as being a complex mixture containing enzymes responsible for the process we now call glycolysis, and over the next half-century all the metabolites and enzymes of glycolysis were described. This classic biochemistry of using a cellfree extract to catalyse a biological process has formed the basis of experiments that have occupied many of my research efforts over the past 30 years. One feature of Buchner’s experiments was that cell-free systems never produced anything like the conversion of sugars to alcohol that whole yeast cells can achieve. Even his very concentrated extract, equivalent to a thick slurry of yeast cells, produced carbon dioxide and alcohol at a rather slow rate, which we can retrospectively estimate as less than 1% of the potential enzyme activity. Typically in the production of wine, the starting liquor contains about 20% by weight of sugars, which are converted completely to ethanol, CO2, yeast biomass and the all-important minor flavour components. But with the early experiments with yeast extracts, only a small amount of the added sugar could be converted to ethanol before the process stopped after a day or two. One of the principal reasons is that the control of glucose entry to the glycolytic pathway is, at least in part, provided by the yeast cell membrane, which of course is lost in making the cellfree extracts. We can now appreciate that the restrained metabolism was due mainly to uncontrolled phosphorylation of glucose, and the

D-Xylulokinase (ATP: D-xylulose 5-phosphotransferase EC 2.7.1.17) was purified from a newly isolated thermophilic Saccharococcus caldoxylosilyticus in which the enzyme is constitutively expressed. The purified enzyme had a specific activity of 60 U/mg at 25°C, and 185 U/mg in its optimum temperature range of between 65 and 75°C. Its Km for xylulose was 0.09 mM at 25°C, and for MgATP 0.16 mM. The molecular mass of the monomer was estimated to be 54 kDa, the holoenzyme comprising two subunits. Stability studies showed that the enzyme was stable up to 65°C, but denatured rapidly at 75°C in Tris buffer. However, it was stabilised by xylulose, which accounts for its high optimum temperature for activity. N-terminal amino acid analysis produced the sequence DHVIGVDLGTSAVKALLVD … which has 65% identity with two other Bacillus xylulokinases as deduced from their gene sequences, and somewhat less identity with other known xylulokinase sequences.

AbstractEnzyme activity refers to the general catalytic properties of an enzyme, and enzyme assays are standardized procedures for measuring the amounts of specific enzymes in a sample.

We examine the ligand requirements for the divinylsulphone (DVS) based T-gel to bind immunoglobulins, The original gel consisted of 2-mercaptoethanol coupled to a DVS activated support, with both the thioether and sulphone sulphurs thought necessary for protein binding. No differences in the capacity for human IgG were observed for a highly activated gel coupled with mercaptoethanol, or when the same activated gel was incubated at high pH to hydrolyse the majority of its reactive groups before the remainder were coupled with the thiol, indicating that the thioether S may be replaced with a hydroxyl O. Increasing the time of the DVS-activation results in gels with higher concentrations of immobilised sulphone but lower concentrations of active groups, The IgG capacities of the mercaptoethanol coupled gels were found to increase with the time of the activation reaction, which may be exploited to produce high capacity gels while minimising the concentration of DVS, Reducing the vinyl of the DVS-activated gel with borohydride was found to decrease the amount of protein binding, with residual binding being attributed to the presence of hydrolysed or crosslinked sulphones in the gel. Reacting the activated gels with amines decreased the capacity for IgG still further, suggesting that not only are these ligands unable to bind IgG, they also prevent its interacting with neighbouring sulphones, perhaps due to the small amount of positive charge they carry. (C) 1998 John Wiley & Sons, Ltd.

Publisher Summary The chapter presents a study on chaperonins from Thermoanaerobacter species. A notable difference between the Escherichia coli GroE proteins and the Thermus chaperonins is that whereas the former interact only in the presence of ATP, the latter interact enough strongly to be isolated together as a complex (cpn60) 14 (cpnl0) 7 . Thermus species are aerobic bacteria, found free living in many hot water environments, there are many anaerobic thermophilic bacteria occurring in more sheltered unaerated parts of hot springs. Of the most common are strains of Thermoanaerobacter , one of which, Thermoanaerobacter thermohydrosulfuricus , often makes up more than 90% of the microbial biomass in these environments. On the basis of its ability to form spores it was originally called Clostridium thermohydrosulfuricum , which has now been reclassified along with a number of other organisms in the Thermoanaerobacter genus. In addition, the much studied and closely related strain of this species Thermoanaerobium brockii, which has been reclassified as Thermoanaerobacter brockii is the presented in the chapter.

Chaperone proteins assist in the folding of some newly synthesized proteins and inhibit protein aggregation. The Thermoanaerobacter brockii chaperonin proteins (Tbr-EL and Tbr-ES) have recently been purified and characterized [Truscott, W.N., Høj, P. B., & Scopes, R. K. (1994) Eur. J. Biochem. 222, 277-284]; Tbr-EL was a single seven-membered toroid, unlike most GroELs which exist as double toroids. Using high-resolution gel filtration chromatography, we have resolved the purified Tbr-EL into single ringed (Tbr-EL7) and double ringed (Tbr-EL14) species. The latter contained tightly bound Tbr-ES co-chaperonin (Tbr-EL14.Tbr-ES7). In the presence of Mg.ATP and either Escherichia coli GroES (Eco-ES) or Tbr-ES (i.e., under protein folding conditions), the isolated Tbr-EL7 rapidly dimerized to the Tbr-EL14.Eco-ES7 or Tbr-EL14.Tbr-ES7 complexes. The doubly toroidal species thus formed contained > or = 6 molecules tightly bound ADP and one GroES7 and are similar to the asymmetric chaperonin complex isolated from Thermus thermophilus [Taguch, H., Konishi, J., Ishii, N., & Yoshida, M. (1991) J. Biol. Chem. 266, 22411-22418]. The isolated Tbr-EL7 and Tbr-EL14.Tbr-ES7 hydrolyzed ATP at approximate to 2 and 1 min-1, respectively. Addition of a molar excess of Eco-ES7 to the isolated Tbr-EL7 reduced the ATPase activity to 1 min-1, consistent with the formation of Tbr-EL14.Eco-ES7. Eco-ES7 failed to inhibit the Tbr-El14.Tbr-ES7 complex. The isolated Tbr-EL14.Tbr-ES7 complex did not support the folding of Rubisco under nonpermissive conditions. Only when the complex was supplemental with additional GroES was folding of Rubisco observed; i.e., one molar equivalent of GroES was not sufficient for folding. Both Tbr-EL7 and Tbr-EL14.Tbr-ES7 bound on unfolded [35S] Rhodospirillum rubrum Rubisco per mole particle. In contrast, Eco-EL14 bound 2 mol of protein per mole particle, consistent with each toroid having a peptide binding site. Eco-EL14.Eco-ES7 complex only bound one unfolded protein, thus GroES binding blocks one GroEL peptide binding site. Addition of Eco-ES7 to a Eco-EL14.Rubisco2 complex did not result in the displacement of one molecule of Rubisco but in the formation of a ternary Eco-EL14.Rubisco2.Eco-ES7 complex.

A number of enzymes that are used in clinical analysis have been studied in relation to the effect of temperature on their activity. Both V-max and K-m were determined over a temperature range from 13 to 55 degrees C. Whereas V-max values increased steadily until denaturation point with all enzymes, the effect of temperature on K-m was more variable. With most enzymes there was a gradual increase in K-m, often with a sharp rise close to the denaturation temperature. In most cases, K-m did not increase as fast as V-max, consequently the enzyme efficiency, V-max/K-m, also increased slightly with temperature. However this was not the case with rabbit muscle lactate dehydrogenase, for which the K-m for pyruvate increased faster than V-max. As a consequence, it was predicted and confirmed that enzymatic analysis of pyruvate using lactate dehydrogenase is more rapid at 20 degrees C than at 35 degrees C or 50 degrees C.