The long-range film structure of 15 base pair, thiol-derivatized duplexes tethered through single 3′ and 5′ linkages to a Au(111) surface is measured via scanning probe microscopy. Film morphology of the 3′ linked duplexes differs from that of its 5′ counterpart as observed by scanning probe microscopy in contact mode under buffer solution. No structural features are distinguished in a film formed with the 5′ linked duplexes as measured with either a bare silicon nitride or chemically modified probe. However, a distinct pattern in the film structure of the 3′ linked duplexes is measured with a chemically modified, hydrophobic tip. Small changes in linker orientation can greatly affect DNA film structure, and a hypothesis for the self-assembly mechanism of the 3′ linked duplexes is presented. Factors such as linker placement and composition must be considered in comparing DNA microarrays formed with different tethering methods.

Bis(peroxo)vanadium(V) complexes are widely investigated as anticancer agents. They exert their antitumor and cyctotoxic effects through inhibition of tyrosine phosphatases and DNA cleavage, respectively. The latter process remains poorly understood. The mechanism of DNA cleavage by NH(4)[(phen)V(O)(eta(2)-O(2))(2)] (phen = 1,10-phenanthroline) was investigated. Kinetic studies on DNA cleavage revealed that the complex is a single-strand nicking agent with no specificity. EPR experiments using 2,2,6,6-tetramethyl-4-piperidone (TMP) and 5,5'-dimethyl-1-pyrroline-N-oxide (DMPO) as spin-traps for singlet oxygen and hydroxyl radical, respectively, implicated hydroxyl radical production upon photodecomposition of bis(peroxo)vanadium(V). This was corroborated by benzoate inhibition of DNA strand scission and stoichiometric oxidation of 2-propanol to acetone upon irradiation of bis(peroxo)vanadium(V) phenanthroline. High-resolution polyacrylamide gel analysis of the vanadium cleavage reaction and [Fe(II)EDTA](2)(-)/H(2)O(2) resulted in comigration of "ladder" pattern bands, which superimposed when both reactions were run on the same lane. These findings identify hydroxyl radical produced from the photooxidation of the peroxo ligand on vanadium as the active species in DNA cleavage.

Phenylalanine hydroxylase converts phenylalanine to tyrosine utilizing molecular oxygen and tetrahydropterin as a cofactor, and belongs to the aromatic amino acid hydroxylases family. The catalytic domains of these enzymes are structurally similar. According to recent crystallographic studies, residue Tyr179 in Chromobacterium violaceum phenylalanine hydroxylase is located in the active site and its hydroxyl oxygen is 5.1 A from the iron, where it has been suggested to play a role in positioning the pterin cofactor. To determine the catalytic role of this residue, the point mutants Y179F and Y179A of phenylalanine hydroxylase were prepared and characterized. Both mutants displayed comparable stability and metal binding to the native enzyme, as determined by their melting temperatures in the presence and absence of iron. The catalytic activity ( k(cat)) of the Y179F and Y179A proteins was lower than wild-type phenylalanine hydroxylase by an order of magnitude, suggesting that the hydroxyl group of Tyr179 plays a role in the rate-determining step in catalysis. The K(M) values for different tetrahydropterin cofactors and phenylalanine were decreased by a factor of 3-4 in the Y179F mutant. However, the K(M) values for different pterin cofactors were slightly higher in the Y179A mutant than those measured for the wild-type enzyme, and, more significantly, the K(M) value for phenylalanine was increased by 10-fold in the Y179A mutant. By the criterion of k(cat)/ K(Phe), the Y179F and Y179A mutants display 10% and 1%, respectively, of the activity of wild-type phenylalanine hydroxylase. These results are consistent with Tyr179 having a pronounced role in binding phenylalanine but a secondary effect in the formation of the hydroxylating species. In conjunction with recent crystallographic analyses of a ternary complex of phenylalanine hydroxylase, the reported findings establish that Tyr179 is essential in maintaining the catalytic integrity and phenylalanine binding of the enzyme via indirect interactions with the substrate, phenylalanine. A model that accounts for the role of Tyr179 in binding phenylalanine is proposed.