Effect of polyamines on palmitoyl-coenzyme A-caused inhibition of glucose-6-phosphate dehydrogenase from baker's yeast

Aliphatic polyamines reversed the inhibition of baker's yeast glucose-6-phosphate dehydrogenase by palmitoyl-CoA. The reversal effects of the polyamines on the inhibition were related not only to the number of amino (imino) groups but also to the carbon chain length of polyamine. The relieving constant (K_r) was calculated to determine the absolute concentration of each examined polyamine required to relieve the enzyme from the inhibition. A smaller K_r value indicates a stronger relieving activity. An ethylamine derivative with a large number of amino groups was more effective in reversing the palmitoyl-CoA inhibition. The K_r values of long-chain aliphatic diamines, triamines, and tetramines were smaller than those of short-chain ones. However, n-butylamine and ornithine, composed of four carbons like putrescine, had markedly larger K_r values than putrescine. Substitution of one or two amino groups by carboxyl or hydroxyl groups also appeared to relieve the enzyme from the inhibition. These results suggest that the reversal effect on palmitoyl-CoA inhibition is one of the important roles of polyamines. Furthermore, it is possible that the interaction of palmitoyl-CoA and polyamines physiologically regulates the pentose monophosphate cycle in baker's yeast.     

In vitro regulation of mammary glucose-6-phosphate dehydrogenase activity by palmitoyl coenzyme A, acetate, and polyamines

An in vitro study was conducted to determine whether bovine mammary glucose-6-phosphate dehydrogenase (G6PD) activity was regulated by palmitoyl coenzyme A (CoA), acetate, spermidine, and putrescine and whether these effects were dependent upon stage of lactation. Early lactation explants incubated in media containing palmitoyl CoA or acetate had reduced (P less than 0.01) G6PD activity compared with incubated control explants. G6PD activity in early lactation explants was reduced (P less than 0.05) when incubated with 5 microM palmitoyl CoA or 1 mM acetate compared with 25 microM palmitoyl CoA or 10 mM acetate. Spermidine (0.4 mM) reversed (P less than 0.05) palmitoyl CoA-induced inhibition of early lactation G6PD activity at 5 microM, but not at 25 microM palmitoyl CoA. G6PD activity in early lactation explants was decreased (P less than 0.05) when treated with putrescine (0.4 mM) compared with explants treated with spermidine. Addition of acetate in combination with 5 microM palmitoyl CoA reversed G6PD inhibition (P less than 0.05 for 1 mM and P less than 0.01 for 10 mM) while addition of either level of acetate in combination with 25 microM palmitoyl CoA failed to reverse G6PD inhibition. G6PD activity was higher (P less than 0.01) in early lactation than mid-lactation explants. No statistical differences (P greater than 0.1) were found among any treatments in explants from mid-lactation cows. We conclude that palmitoyl CoA and acetate will inhibit G6PD activity in early lactation, but not mid-lactation explants; addition of spermidine will reverse this inhibition.     

Enhanced proteolysis of glucose-6-phosphate dehydrogenase in the presence of palmitoyl coenzyme A

Palmitoyl coenzyme A at concentrations below its critical micelle concentration increases the rate of proteolysis of baker's yeast glucose-6-phosphate dehydrogenase by proteinase A in the pH range 4-5. both glucose-6-phosphate and NADP protect glucose-6-phosphate dehydrogenase against proteolysis, but these protective effects are diminished in the presence of palmitoyl coenzyme A. Since palmitoyl coenzyme A is known to dissociate glucose-6-phosphate dehydrogenase into dimers, the results imply that the in vivo half life of glucose-6-phosphate dehydrogenase may be controlled by a process based on the regulation of the oligomeric structure of the enzyme by the collective actions of various molecules, including palmitoyl coenzyme A.     

Purification of a novel coenzyme F420-dependent glucose-6-phosphate dehydrogenase from Mycobacterium smegmatis.

A variety of Mycobacterium species contained the 5-deazaflavin coenzyme known as F420. Mycobacterium smegmatis was found to have a glucose-6-phosphate dehydrogenase that was dependent on F420 as an electron acceptor and which did not utilize NAD or NADP. The enzyme was purified by ammonium sulfate fractionation, phenyl-Sepharose column chromatography, F420-ether-linked aminohexyl-Sepharose 4B affinity chromatography, and quaternary aminoethyl-Sephadex column chromatography, and the sequence of the first 26 N-terminal amino acids has been determined. The response of enzyme activity to a range of pHs revealed a two-peak pattern, with maxima at pH 5.5 and 8.0. The apparent Km values for F420 and glucose-6-phosphate were, respectively, 0.004 and 1.6 mM. The apparent native and subunit molecular masses were 78,000 and approximately 40,000 Da, respectively.     

A kinetic study of glucose-6-phosphate dehydrogenase.

The steady state kinetics of pig liver glucose-6-phosphate dehydrogenase is consistent with an ordered, sequential mechanism in which NADP is bound first and NADPH released last. Kia is 9.0 muM, Ka is 4.8 muM, and Kb is 36 muM. Glucosamine 6-phosphate, a substrate analogue and competitive inhibitor, is used to help rule out a possible random mechanism. ADP is seen to form a complex with the free form of the enzyme whereas ATP forms a complex with both the free and E-NADP forms of the enzyme. The KI for the E-ADP complex is 1.9 mM, while the Ki values for the E-ATP and E-NADP-ATP complexes are 7.2 and 4.5 mM, respectively.     

Hyperanodic forms of human glucose-6-phosphate dehydrogenase.

Pure glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate:NADP+ 1-oxidoreductase, EC 1.1.1.49) is transformed into 'hyperanodic forms' when incubated at acidic pH and in the presence of NADP+ with excess of glucose-6-phosphate or with some 'NADP+ modifying proteins' purified from the same cells. The enzyme hyperanodic forms exhibit low isoelectric point, altered kinetic properties and high lability to heat, urea, and proteolysis. Differences between hyperanodic and native forms of glucose-6-phosphate dehydrogenase are also noted by microcomplement fixation analysis, ultraviolet absorbance difference spectrum and fluorescence emission spectrum. Drastic denaturation of the enzyme by urea and acid treatment did not suppress the difference of isoelectric point between native and hyperanodic forms of glucose-6-phosphate dehydrogenase. From our data we suggest that the conversion into hyperanodic forms could be due to the covalent binding on the enzyme of a degradation product of the pyridine nucleotide coenzyme. This modification could constitute a physiological transient step toward the definitive degradation of the enzyme.