Neocarzinostatin chromophore. Assignment of spectral properties and structural requirements for binding to DNA.
The site responsible for the mercaptan (or borohydride)-stimulated DNA scission activity of neocarzinostatin chromophore (NCS-Chrom) is located in the central C12-subunit of the molecule. This has been determined by studies of the characteristic spectral properties of the chromophore and its reduction products and of the spectral changes induced by their interaction with its apoprotein (apo-NCS) and DNA. The UV-visible absorption, fluorescence, CD, and MCD spectral properties of the major nonprotein chromophoric component of neocarzinostatin (NCS-Chrom A) are assigned to its component substructures, the 2-hydroxy-5-methoxy-7-methyl-1-naphthoate, the five-membered cyclic carbonate ring (1,3-dioxolan-2-one), the 2,6-dideoxy-2-methylaminogalactose, and the incompletely defined C12-subunit which links the other three residues. Although the major source of its UV-visible absorption is the naphthoic acid residue (HNA-NCS), a significant absorption from approximately 260 to 330 nm is due to the presence of the highly unsaturated C12-subunit. The presence of the C12-subunit and, to a lesser extent, the cyclic carbonate reduces the intensity of the fluorescence emission of the fluorophore of NCS-Chrom A, the HAN-NCS subunit. The CD activity of NCS-Chrom A is also due to the presence of the C12-subunit. The MCD activity of NCS-Chrom A, however, is completely accounted accounted for by the naphthoic acid residue. A role for the C12-subunit in the binding of NCS-Chrom to DNA or apo-NCS is indicated by the resultant absorption hypochromicity in the region assigned to the C12-moiety. Limited modification of the C12-subunit (by mercaptan or borohydride) inactivates the chromophore for DNA strand scission, although both products still bind DNA. The naphthoic acid residue alone is not sufficient for binding to DNA. Therefore, the intercalation of the naphthoate residue between DNA base pairs requires the binding of NCS-Chrom to DNA probably via electrostatic interaction between the positively charged 2-methylamino group of the galactose residue and the negatively charged oxygens of the phosphate in the DNA backbone. The C12-unit is viewed as forming a short-lived reduction-activated species that, in the presence of oxygen, causes single-strand breaks in DNA. The cyclic carbonate residue is not required for in vitro DNA strand scission activity but affects its stability with respect to hydrolysis and reactivity with mercaptan. If DNA is absent the activated species decompose to inactive products, including a mercaptan (or hydrogen) addition product of the C12-subunit.
D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000378:metabolism*; D002417:Cattle; D002942:Circular Dichroism; D004247:DNA / Q000378:metabolism*; D053281:Enediynes; D006863:Hydrogen-Ion Concentration; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013864:Thioglycolates / Q000378:metabolism; D009353:Zinostatin / Q000031:analogs & derivatives / Q000378:metabolism*
D000903:Antibiotics, Antineoplastic; D053281:Enediynes; D013438:Sulfhydryl Compounds; D013864:Thioglycolates; C005231:methylmercaptan; C032372:neocarzinostatin chromophore; D004247:DNA; D009353:Zinostatin; C037474:methyl thioglycolate