Inactivation of pyruvate decarboxylase by 3-hydroxypyruvate.

Pyruvate decarboxylase from Zymomonas mobilis is inhibited by 3-hydroxypyruvate, which can also act as a poor substrate. While catalysing the decarboxylation of this alternative substrate, the enzyme undergoes a progressive but partial inactivation over several hours. The extent of inactivation depends upon the pH and upon the concentration of 3-hydroxypyruvate. After partial inactivation and removal of unchanged 3-hydroxypyruvate, enzymic activity recovers slowly. We suggest that inactivation results from accumulation of enzyme-bound glycollaldehyde, which is relatively stable, possibly because it is dehydrated to form an acetyl group.     

Inactivation of rat gastric mucosal histidine decarboxylase by phosphatase

Histidine decarboxylase of supernatants as well as of purified preparations from rat gastric mucosa is inactivated by a non-specific phosphatase in the absence of pyridoxal 5'-phosphate. The inactivation is a time and concentration-dependent process. Pyridoxal 5'-phosphate, but not histidine, protects the enzyme against phosphatase action. The inactivation is reversible, only pyridoxal 5'-phosphate reactivates the inactivated enzyme. Pyridoxamine 5'-phosphate is ineffective for histidine decarboxylase, but is converted into an active coenzyme only in gastric supernatant. Evidence for the occurrence of an active phosphatase in gastric tissue is also presented; its properties are those of an acid phosphatase and are similar to those of phosphatases hydrolyzing pyridoxal 5'-phosphate in other tissues. The data indicate that phosphatase promotes apoenzyme formation and may play a role in the regulation of histamine synthesis.     

Inactivation of yeast ornithine decarboxylase by polyamines in vivo does not result from the incorporation of polyamines into enzyme protein

The peptide mixture obtained from controlled proteolytic digestion of ligandin with proteinase K or subtilisin retained 40% of glutathione-S-transferase and steroid isomerase activities, immunological reactivity and lower affinity bilirubin binding but binding at the primary site was abolished. When these limited proteolytic digests, which had no intact ligandin as determined by SDS gel electrophoresis, were subjected to Sephadex G-75 column chromatography, 40–50% of the peptide fragments were recovered in fractions where intact ligandin eluted. The results suggest that intact ligandin is not required for enzymatic activities, binding of bilirubin at the secondary site, or immunological reactivity; steroid isomerase and glutathione-S-transferase activities are modulated in a parallel manner and may be mediated by the same region of the protein, and primary and secondary binding sites for bilirubin are distinct and independent, despite nicks introduced by proteolysis in ligandin's subunits, some of the fragments remain associated under non-denaturing conditions and the susceptibility of the two subunits to the proteases is different.     

Inactivation of yeast hexokinase B by triethyltin bromide

Triethyltin bromide was found to demonstrate temperature-dependent inactivation of yeast hexokinase B. At temperatures of 20 degrees C or lower, little or no inactivation of the enzyme was detected after 2 h of reaction with 50-300 microM concentrations of the reagent. However, incubation at 25 degrees C or higher resulted in an increased rate and extent of loss of the enzyme activity with increasing incubation temperatures. The Arrhenius plot for the inactivation process showed a sharp break at approximately 30 degrees C, with a heat of activation (delta H*) above this temperature of 55.2 kcal, indicating that a triethyltin-induced conformational change occurred at the elevated temperatures. Sugar substrates provided protection against the inactivating effect by reducing the binding of triethyltin to the enzyme. In the absence of glucose, two sites of different affinity for triethyltin exist in the hexokinase monomer. Binding of triethyltin to the enzyme shifted its monomer-dimer equilibrium toward the monomeric form in an early stage of the interaction. Inactivation of the enzyme was associated with a slower subsequent event. Comparative effects of various organotin compounds on the activity of the enzyme indicated that inhibitory potency was associated with increasing hydrophobicity of the alkyl groups attached to the tin.     

Inactivation of mevalonate 5-diphosphate decarboxylase by phosphorothioate analogues of ATP

Mevalonate 5-diphosphate decarboxylase is inactivated by several nucleoside phosphorothioates and the order of effectiveness as inactivators is: (Rp) ATP beta S greater than (Sp) ATP beta S approximately equal to ADP beta S approximately equal to AMPS greater than ATP gamma S. Mevalonate 5-diphosphate protects the enzyme against inactivation and, to a lower extent, so does ATP. As dithiothreitol prevents the enzyme inactivation, these results are interpreted as being the consequence of the interaction of the above mentioned analogues with functional thiol groups of the enzyme.     

Dissociation of yeast 80 S ribosomes by chaotropic salts

Exposure of yeast 80 S ribosomes to chaotropic salts such as NaClO₄ or NaSCN at concentrations as low as 0.4 M resulted in complete dissociation and subsequent aggregation of the ribosomal proteins. However, under similar conditions, both NaCl and NaBr did not cause dissociation and aggregation. The protein precipitate obtained by exposing the ribosomes to 0.5 M NaClO₄ was free of any rRNA contamination as judged by ultraviolet-absorption analysis. Comparison of the two-dimensional polyacrylamide gel electrophoretic analysis of the above ribosomal protein precipitate with that ribosomal proteins isolated by the standard acetic acid extraction procedure revealed that the protein precipitate contained all the ribosomal proteins. Based on these results, a simple method for the isolation of total ribosomal proteins and rRNA under mild, nondenaturing conditions is proposed. A possible mechanism for the dissociation of proteins from the ribosome by chaotropic salts is also discussed.     

Inactivation of bakers' yeast glucose-6-phosphate dehydrogenase by aluminum

Preincubation of yeast glucose-6-phosphate dehydrogenase (G6PD) with Al(III) produced an inactive enzyme containing 1 mol of Al(III)/mol of enzyme subunit. None of the enzyme-bound Al(III) was dissociated by dialysis against 10 mM Tris-HCl, pH 7.0, containing 0.2 mM EDTA at 4 degrees C for 24 h. Citrate, NADP+, EDTA, or NaF protected the enzyme against the Al(III) inactivation. The Al-(III)-inactivated enzyme, however, was completely reactivated only by citrate and NaF. The dissociation constant for the enzyme-aluminum complex was calculated to be 4 x 10(-6)M with NaF, a known reversible chelator for aluminum. Modification of histidine and lysine residues of the enzyme with diethyl pyrocarbonate and acetylsalicylic acid, respectively, inactivated the enzyme. However, the modified enzyme still bound 1 mol of Al(III)/mol of enzyme subunit. Circular dichroism studies showed that the binding of Al(III) to the enzyme induced a decrease in alpha-helix and beta-sheet and an increase in random coil. Therefore, it is suggested that inactivation of G6PD by Al(III) is due to the conformational change induced by Al(III) binding.     

Inactivation of yeast enolase with tetranitromethane

Yeast enolase is inactivated by tetranitromethane with production of 1.2 moles of nitrotyrosine per subunit. Protection is afforded by “conformational” metal ion alone. Enzyme thus inactivated no longer appears to bind “conformational” metal ion. There is evidence against direct coordination of the tyrosine to “conformational” metal ion, suggesting modification of the tyrosyl may obstruct the binding site.     

Inactivation of malonate semialdehyde decarboxylase by 3-halopropiolates: evidence for hydratase activity

Malonate semialdehyde decarboxylase (MSAD) from Pseudomonas pavonaceae 170 catalyzes the metal ion-independent decarboxylation of malonate semialdehyde and represents one of three known enzymatic activities in the tautomerase superfamily. The characterized members of this superfamily are structurally homologous proteins that share a beta-alpha-beta fold and a catalytic amino-terminal proline. Sequence analysis, chemical labeling studies, site-directed mutagenesis, and NMR studies of MSAD identified Pro-1 as a key active site residue in which the amino group has a pKa value of 9.2. The available evidence suggests a mechanism involving polarization of the C-3 carbonyl group of malonate semialdehyde by the cationic Pro-1. A second critical active site residue, Arg-75, could assist in the reaction by placing the substrate's carboxylate group in a favorable conformation for decarboxylation. In addition to the decarboxylase activity, MSAD has a hydratase activity as demonstrated by the MSAD-catalyzed conversion of 2-oxo-3-pentynoate to acetopyruvate. In view of this activity, MSAD was incubated with 3-bromo- and 3-chloropropiolate, and the subsequent reactions were characterized. Both compounds result in the irreversible inactivation of MSAD, making them the first identified inhibitors of MSAD. Inactivation by 3-chloropropiolate occurs in a time- and concentration-dependent manner and is due to the covalent modification of Pro-1. The proposed mechanism for inactivation involves the initial hydration of the 3-halopropiolate followed by a rearrangement to an alkylating agent, either an acyl halide or a ketene. The results provide additional evidence for the hydratase activity of MSAD and further support for the hypothesis that MSAD and trans-3-chloroacrylic acid dehalogenase, the preceding enzyme in the trans-1,3-dichloropropene catabolic pathway, diverged from a common ancestor but conserved the necessary catalytic machinery for the conjugate addition of water.     

Inactivation of yeast enzymes by proteinase A and B and carboxypeptidase Y from yeast.

Changes in the activities of 15 different enzymes during incubation of a crude yeast extract with the purified yeast proteinases A and B, and carboxypeptidase Y, respectively, have been measured. The spectrum of action of the three proteinases on the enzymes measured differs significantly, increasing or decreasing their activities or having no effect. Incubation of purified cytoplasmic malate dehydrogenase or purified mitochondrial malate dehydrogenase with proteinases A and B results in selective inactivation of the cytoplasmic enzyme, whereas the mitochondrial activity is not affected. Carboxypeptidase Y has no effect on the activity of either dehydrogenase. The results support the idea of selective proteolysis as the mechanism of the earlier observed inactivation of cytoplasmic malate dehydrogenase, initiated by the addition of glucose to intact yeast cells grown on acetate as carbon source ("glucose effect").     

Yeast antizyme mediates degradation of yeast ornithine decarboxylase by yeast but not by mammalian proteasome: new insights on yeast antizyme

Mammalian antizyme (mAz) is a central element of a feedback circuit regulating cellular polyamines by accelerating ornithine decarboxylase (ODC) degradation and inhibiting polyamine uptake. Although yeast antizyme (yAz) stimulates the degradation of yeast ODC (yODC), we show here that it has only a minor effect on polyamine uptake by yeast cells. A segment of yODC that parallels the Az binding segment of mammalian ODC (mODC) is required for its binding to yAz. Although demonstrating minimal homology to mAz, our results suggest that yAz stimulates yODC degradation via a similar mechanism of action. We demonstrate that interaction with yAz provokes degradation of yODC by yeast but not by mammalian proteasomes. This differential recognition may serve as a tool for investigating proteasome functions.     

Regulated degradation of yeast ornithine decarboxylase.

Ornithine decarboxylase (ODC) declines in cells that accumulate an excess of polyamines, the downstream products of the enzyme. Superfluous production of polyamines is thus prevented. In animal cells, polyamines reduce ODC activity by accelerating its degradation. Similar down-regulation of ODC activity has been observed in the budding yeast Saccharomyces cerevisiae, but induced degradation has not been documented. Here we show using pulse-chase analysis that the loss of enzyme activity is the result of increased degradation of ODC. Polyamines reduce the half-life of the newly synthesized protein from 3 h to approximately 10 min. Degradation of bulk ODC pools is also accelerated by polyamines, but the absolute rate of turnover is slower, with a half-life of 5 h in untreated and 1 h in treated cells. Newly synthesized ODC polypeptide thus undergoes a process of maturation that renders it relatively resistant to both basal and polyamine-induced degradation. Proteasome mutants have a blunted or absent regulatory response, implicating both the core protease and the regulatory cap of the proteasome in induced degradation of yeast ODC.     

Synthesis, structure, and unprecedented solubility of lipophilic borate salts

The multi-gram scale preparation of halide free lipophilic borate salts from inexpensive precursor compounds 3,3′,5,5′-tetra-tert-butyl-2,2′-biphenol and tetrahydridoboranate salts is reported. The so-called “bortebate” anion is more stable against water and bases than its aluminum analog “altebate”, but bortebate formation is significantly slower. Bortebate salts are highly soluble in hydrocarbon solvents, e.g. >35 mmol/L lithium bortebate in pentane at 20 °C. Quantitative salt metathesis reactions between sodium bortebate and halide salts can be easily achieved by precipitation of the sodium halide in methylene chloride.