PubMed · 7869569
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1994. Propylene.. https://pubmed.ncbi.nlm.nih.gov/7869569/
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Rat glutathione transferase (GST) 8-8 displays high catalytic activity with alpha, beta-unsaturated carbonyl compounds, including lipid peroxidation products such as 4-hydroxyalkenals. The catalytic efficiency of the related class Alpha GST 1-1 is substantially lower with the same substrates. Chimeric enzymes were prepared by replacing N-terminal subunit 8 segments of different lengths (6, 25, or 100 residues) with corresponding sequences from subunit 1 using recombinant DNA techniques. The chimeric subunit r1(25)r8, containing 25 amino acid residues from subunit 1, had the same low activity with alkenal substrates as that displayed by subunit 1. Mutation of Ala-12 into Gly in r1(25)r8 gave rise to the high alkenal activity characteristic of subunit 8, showing the importance of amino acid residue 12 for the activity. However, other structural determinants are also essential, as demonstrated by the corresponding Ala-12-->Gly mutation in subunit 1, which did not afford high alkenal activity. The results show that a single point mutation in a GST subunit may give rise to a 100-fold increase in catalytic efficiency with certain substrates. Introduction of such mutations may have contributed to the biological evolution of GST isoenzymes with altered substrate specificities and may also find use in the engineering of GSTs for novel functions.
The first step in the biosynthesis of taxol in Pacific yew (Taxus brevifolia) is the cyclization of the universal diterpene precursor geranylgeranyl pyrophosphate to taxa-4(5),11(12)-diene. This parent olefin of the taxane diterpenoids is then elaborated to taxol and related compounds by a complex series of reactions involving oxidations and side-chain acylations. Cyclization activity is located principally in yew stem bark and adhering cambium. The operationally soluble cyclization enzyme was partially purified (approximately 600-fold) by combination of anion exchange, hydrophobic interaction, and dye-ligand chromatography. Nondenaturing, followed by denaturing, polyacrylamide gel electrophoresis, in combination with gel permeation chromatography, allowed the identification of taxadiene synthase as a monomeric protein of molecular weight 79,000. In general properties (divalent metal ion requirement, kinetic constants, molecular weight), the taxadiene synthase of Pacific yew is similar to the diterpene cyclase abietadiene synthase involved in resin acid biosynthesis in other gymnosperms. However, in pH optimum and response to inhibitors, these two diterpene cyclases are distinctly different. The activity (and enzyme protein) levels of Pacific yew taxadiene synthase are much lower than those for abietadiene synthase of lodgepole pine stem (constitutive) or of grand fir stem (wound-inducible) and the enzyme is not inducible to higher levels by stem wounding or elicitor treatment.
A double-stranded DNA cleavage mechanism by a novel enediyne type antitumor antibiotic, dynemicin A, has been investigated through sequence-dependent strand breakage of a series of duplexes containing a single nucleotide gap. We found that (1) dynemicin A breaks specifically at the 3'-shifted position by one base opposite the gap, (2) the strong cleavage is detected at 5'-Pu_Pu/3'-PyPuPy sequences, and (3) dynemicin H (aromatized form of dynemicin A) gives only a small inhibition effect (20%) on the cleavage of gapped duplex by dynemicin A. The long half-life of aromatization of dynemicin A (118 min, in the presence of DNA) obtained from HPLC analysis provides enough time for the second cleavage. The present results strongly indicate a two-step mechanism for the double-stranded DNA scission of dynemicin A. Namely, this double-stranded break is caused by two drug molecules, each of which cuts one DNA strand.