Studies on the mechanism of sheep liver cytosolic aldehyde dehydrogenase. The effect of pH on the aldehyde binding reactions and a re-examination of the problem of the site of proton release in the mechanism.

Initial-rate measurements and stopped-flow spectrophotometric experiments over a wide range of pH implicate an enzyme group of pKa approximately 6.6 affecting the aldehyde binding reactions. It is possible, though not proved, that the group involved is the cysteine residue involved in catalysis. Stopped-flow fluorescence studies show that a group of pKa greater than 8.5 facilitates hydrolysis of the NADH-containing acyl-enzyme species. The identity of this group is quite unknown. Studies with 4-nitrobenzaldehyde show that this substrate gives marked substrate inhibition at quite low (less than 20 microM) concentrations. The mechanism of catalysis seems to be the same as for propionaldehyde oxidation. It is argued that proton release occurs with both substrates on hydrolysis of the NADH-containing acyl-enzyme and not before hydride transfer, as has been previously suggested [Bennett, Buckley & Blackwell (1982) Biochemistry 21, 4407-4413].     

The effects of Mg2+ on certain steps in the mechanisms of the dehydrogenase and esterase reactions catalysed by sheep liver aldehyde dehydrogenase. Support for the view that dehydrogenase and esterase activities occur at the same site on the enzyme.

Stopped-flow experiments in spectrophotometric and fluorescence modes reveal different aspects of the aldehyde dehydrogenase mechanism. Spectrophotometric experiments show a rapid burst of NADH production whose course is not affected by Mg2+. The slower burst seen in the fluorescence mode is markedly accelerated by Mg2+. It is argued that the fluorescence burst accompanies acyl-enzyme hydrolysis and, therefore, that Mg2+ increases the rate of this process. Experiments on the hydrolysis of p-nitrophenyl propionate indicate that acyl-enzyme hydrolysis is indeed accelerated by Mg2+ and a combination of Mg2+ and NADH. Vmax. values for p-nitrophenyl propionate hydrolysis in the presence of NADH and NADH and Mg2+ agree closely with the specific rates of acyl hydrolysis from the E . NADH . acyl and E . NADH . acyl . Mg2+ complexes seen in the dehydrogenase reaction with propionaldehyde. These observations support the view that esterase and dehydrogenase activities occur at the same site on the enzyme. Other evidence is presented to support this conclusion.     

Studies of sugars and sorbitol in developing corn kernels

Sugars and sorbitol were determined on corn (Zea mays L.) kernels harvested at various developmental stages, using sugary (su), sugary-sugary enhancer (su se), and starchy (Su) cultivars. In all cultivars tested, the sorbitol content increased from trace amounts in unpollinated ovules to a maximum at about the time that rapid starch synthesis was proceeding. Thereafter, sorbitol and sugars decreased continuously to the mature dry stage. Sorbitol in the su se kernels was higher than that of other cultivars from 28 days postpollination onwards; sucrose and maltose were higher from 21 days onwards. [¹⁴C]Sorbitol was recovered from kernel base, pedicel, and endosperm of IL677a (su se) kernels after allowing a flag leaf to fix ¹⁴CO₂ photosynthetically. No [¹⁴C]sorbitol was detected in the shank of the ear, and none was detected by the gas chromatograph. [¹⁴C]Sucrose was the predominant labeled substance recovered from the kernel base, pedicel, and endosperm tissues during the 10-h chase period, as well as from the shank of the ear, and nonradioactive sucrose was the predominant ethanol-soluble compound detected by the gas chromatograph. Hence, sorbitol appears not to be translocated from corn leaves as it is in certain woody plants of the rose family. The altered sugar profile of su se kernels may be related to reduced starch synthesis, but the biochemical mechanism is not yet known.     

Kinetic and physical properties of Co2+ enolase

The activation of yeast enolase by cobaltous ion in 0.1 M KCl is characterized by an activation constant of 1 microM and an inhibition constant of 18 microM. Measurements of binding of Co2+ to the apoenzyme show that a maximum of four Co2+ ions are bound per dimer in the presence or absence of substrate although binding is far tighter in the presence of substrate. Ultraviolet spectral titrations show evidence for a conformational change due exclusively to the binding of the first two ions of Co2+. Both visible and EPR spectra confirm that the environment of the first pair of cobalt ions ("conformational sites") is markedly different from that of the second pair in the "catalytic" sites. Cobalt at the conformational site appears to be a tetragonally distorted octahedral complex while the second pair of metal ions appears to be in a more regular tetrahedral symmetry. Addition of either Mg2+ or substrate to the enzyme with only one pair of cobalt ions per dimer causes striking changes in the metal ion environment. The conformational metal sites appear sufficiently shielded from solvent to be inaccessible to oxidation by H2O2, in contrast to the second pair of cobaltous ions whose ready oxidation by H2O2 inactivates the enzyme. Comparison of kinetic and binding data suggests that only one site of the dimeric enzyme can be active, since activity requires more than two metals bound per dimer and inactivation results from the binding of the fourth ion per dimer.     

A study of the role of the hexose monophosphate pathway with respect to fatty acid biosynthesis in sporulation of Saccharomyces cerevisiae

13C NMR was used to study the pattern of label incorporation from [2-13C]acetate into trehalose during sporulation in Saccharomyces cerevisiae. A wild-type strain and a strain homozygous for the zwf1 mutation (which affects glucose-6-phosphate dehydrogenase) were used. In the wild-type it was possible to deduce the cycling of glucose 6-phosphate around the hexose monophosphate pathway whilst in the mutant strain this did not occur. The requirement of the hexose monophosphate pathway for providing NADPH for fatty acid biosynthesis was examined using 13C NMR and GC/MS. The wild-type strain produced a typical profile of fatty acids with palmitoleic acid being the most abundant whereas the mutant contained only one-quarter the amount of total fatty acid. As zwf1 homozygous diploids are able to sporulate this indicates that the large amount of fatty acid biosynthesis observed in sporulation of wild-type strains is not essential to the process.     

The role of the metal ion in the mechanism of the K+-activated aldehyde dehydrogenase of Saccharomyces cerevisiae.

The effect of K+ on assays of the enzyme was studied and it appears that the activation occurs slowly by a two-step process. Kinetic measurements suggest that the enzyme-catalysed reaction can proceed slowly (0.4%) in the complete absence of K+. The enzyme exhibits a K+-activated esterase activity, which is further activated by NAD+ or NADH. Stopped-flow studies indicated that the principal effect of K+ on the dehydrogenase reaction is to accelerate a step (possibly acyl-enzyme hydrolysis) associated with a fluorescence and small absorbance transient that occurs after hydride transfer and before NADH dissociation from the terminal E-NADH complex. The variation of activity of the enzyme with pH was studied. An enzyme group with pKa approx. 7.1 apparently promotes enzyme activity when in its alkaline form.     

Effect of training/detraining on submaximal exercise responses in humans

Human subjects participated in a training/detraining paradigm which consisted of 7 wk of intense endurance training followed by 3 wk of inactivity. In previously sedentary subjects, training produced a 23.9 +/- 7.2% increase in maximal aerobic power (V02max) (group S). Detraining did not affect group S V02max. In previously trained subjects (group T), the training/detraining paradigm did not affect V02max. In group S, training produced an increase in vastus lateralis muscle citrate synthase (CS) activities (nmol.mg protein-1. min-1) from 67.1 +/- 14.5 to 106.9 +/- 22.0. Detraining produced a decrease in CS activity to 80 +/- 14.6. In group T, pretraining CS activity (139.5 +/- 14.9) did not change in response to training. Detraining, however, produced a decrease in CS activity (121.5 +/- 7.8 to 66.8 +/- 5.9). Group S respiratory exchange ratios obtained during submaximal exercise at 60% V02max (R60) decreased in response to training (1.00 +/- 0.02 to 0.87 +/- 0.02) and increased (0.96 +/- 0.02) after detraining. Group T R60 (0.91 +/- 0.01) was not affected by training but increased (0.89 +/- 0.02 to 0.95 +/- 0.02) after detraining. R60 was correlated to changes in CS activity but was unrelated to changes in V02max. These data support the hypothesis that the mitochondrial content of working skeletal muscle is an important determinant of substrate utilization during submaximal exercise.     

A tissue-specific MAR/SAR DNA-binding protein with unusual binding site recognition.

A human cDNA was cloned that encodes a DNA-binding protein (SATB1) that is expressed predominantly in thymus and binds selectively to the nuclear matrix/scaffold-associating DNAs (MARs/SARs). Missing nucleoside experiments showed that SATB1 selectively binds in a special AT-rich sequence context where one strand consists of mixed A's, T's, and C's, excluding G's (ATC sequences). When this feature is destroyed by mutation, SATB1 binding is greatly reduced even if the direct contact sequence remains intact. Conjunctional SATB1-binding sequences become stably unpaired in supercoiled DNA. Specific mutations that diminish the unwinding potential greatly reduce SATB1 binding. However, SATB1 does not bind single-stranded DNA. Chemical interference assays show that SATB1 binds along the minor groove with very little contact with the bases. This suggests that SATB1 recognizes the ATC sequence indirectly through the altered sugar-phosphate backbone structure present in the double-stranded DNA.     

Long-chain alcohol and aldehyde dehydrogenase activities in Acinetobacter calcoaceticus strain HO1-N.

Three alcohol dehydrogenases have been identified in Acinetobacter calcoaceticus sp. strain HO1-N: an NAD(+)-dependent enzyme and two NADP(+)-dependent enzymes. One of the NADP(+)-dependent alcohol dehydrogenases was partially purified and was specific for long-chain substrates. With tetradecanol as substrate an apparent Km value of 5.2 microM was calculated. This enzyme has a pI of 4.5 and a molecular mass of 144 kDa. All three alcohol dehydrogenases were constitutively expressed. Three aldehyde dehydrogenases were also identified: an NAD(+)-dependent enzyme, an NADP(+)-dependent enzyme and one which was nucleotide independent. The NAD(+)-dependent enzyme represented only 2% of the total activity and was not studied further. The NADP(+)-dependent enzyme was strongly induced by growth of cells on alkanes and was associated with hydrocarbon vesicles. With tetradecanal as substrate an apparent Km value of 0.2 microM was calculated. The nucleotide-independent aldehyde dehydrogenase could use either Würster's Blue or phenazine methosulphate (PMS) as an artificial electron acceptor. This enzyme represents approximately 80% of the total long-chain aldehyde oxidizing activity within the cell when the enzymes were induced by growing the cells on hexadecane. It is particulate but can be solubilized using Triton X-100. The enzyme has an apparent Km of 0.36 mM for decanal.     

13C NMR analysis of a developmental pathway mutation in Saccharomyces cerevisiae reveals a cell derepressed for succinate dehydrogenase.

13C nuclear magnetic resonance (NMR) spectroscopy was used to study the metabolism of [2-13C]acetate in a diploid strain of Saccharomyces cerevisiae homozygous for the spo50 mutation. This mutation results in failure to initiate sporulation and suppresses spd mutations (which cause derepressed sporulation). By analysing the pattern of 13C-labelling in glutamate it was deduced that the glyoxylate cycle is responsible for most of the acetate utilization and that there is very little tricarboxylic acid cycle activity. The labelling of alpha,alpha'-trehalose indicated that gluconeogenesis and the hexose monophosphate pathway operate in a similar way to the wild-type. The mutant strain has higher levels of succinate dehydrogenase than the wild-type. All of the physiological alterations caused by the spo50 mutation can be explained by this difference.