Progress towards synthetic enzymes for phosphoester hydrolysis.

Synthesis of artificial enzymes for catalyzing phosphoester hydrolysis has been attracting interest for a long time. The remarkable discovery that lanthanide ions catalyze the hydrolysis of DNA and RNA spurred the trend. Currently, progress is being made, mainly in the preparation of homogeneous catalysts, the promotion of catalytic activity by using acid/base cooperation within catalysts, the detailed understanding of the reaction mechanisms involved, and the design of artificial enzymes expressing high specificity and catalytic turn-over.

Lanthanide ions for the first non-enzymatic formation of adenosine 3',5'-cyclic monophosphate from adenosine triphosphate under physiological conditions.

Adenosine 3',5'-cyclic monophosphate (cAMP) is formed from adenosine triphosphate at pH 8 and 50 degrees C by use of lanthanide ions. Pr3+ and La3+ are the most active. The cAMP formation is more efficient at higher pH, where the mixture is made homogeneous by the addition of beta-cyclodextrin. The potential functioning of lanthanide ions as the catalytic center of an artificial adenylate cyclase is indicated.

Catalytically active species for CeCl3-induced DNA hydrolysis.

Cerium(III) chloride efficiently hydrolyzes DNA at pH 7 under aerobic conditions. A titration study showed that Ce(III) is oxidized to Ce(IV) in the reaction mixture and the resultant Ce(IV) is responsible for the DNA hydrolysis.

Kinetic studies on Ce(IV)-induced hydrolysis of single-stranded and double-stranded oligonucleotides.

The Ce(IV)-induced hydrolyses of DNA are kinetically investigated. The formation constants of the Ce(IV)-DNA complexes are in the following order: the single-stranded DNA > the double-stranded DNA >> the dinucleotide. On the other hand, the catalytic rate constants for the single-stranded DNA and the double-stranded DNA are comparable with each other, but both of them are much smaller than the value for the dinucleotide hydrolysis.

Enormous catalyses of lanthanide metal ions for unprecedentedly fast hydrolysis of 3',5'-cyclic adenosine monophosphate.

3',5'-cyclic adenosine monophosphate (cAMP) is efficiently hydrolyzed by use of lanthanide metal(III) ions as catalysts. The acceleration by 10(-2) M of Ce(III) is remarkable (more than 10(11) fold) at pH 8.0, 30 degrees C, decreasing the half-life from a half million years to 35 seconds. The catalytic activity is in the following order: Ce >> Pr > Nd, La > Y, Sm, Dy > others. The catalysis is ascribed to cooperation of three or four lanthanide metal ions.

Improvement of efficiency of the Ce(IV)-induced DNA scission--relationship between the kinetic parameters (k(cat) and Km) and the DNA structure.

The Michaelis constant (Km) for double-stranded DNA, single-stranded DNA, and dinucleotide hydrolysis by Ce(IV) ion are 4.4, 15, and more than 40 mM, respectively. The order of the k(cat), however, is dinucleotide >> oligonucleotides. Not only the improvement of k(cat) but also that of Km is important for the design of an efficient artificial nuclease.

Homogeneous catalyst for DNA hydrolysis (4). Efficient DNA hydrolysis by lanthanide--saccharide complexes.

Homogeneous and neutral solutions are prepared by mixing Ce(NH4)2(NO3)6 with either isomaltose, melibiose, gentiobiose, palatinose, mannitol, sorbitol, galactitol, or glucamine in pH 7 hepes buffer ([Ce(IV)]0/[monomeric residue of saccharide]0 = 1). In contrast, amylose, cyclodextrins, maltose, glucose, and fructose provide only heterogeneous mixtures. The homogeneous solution of 1:1 Ce(IV)/glucamine system is active for DNA hydrolysis: the pseudo-first-order rate constant for the hydrolysis of thymidylyl(3'-->5')thymidine at pH 7.0 and 50 degrees C is 0.010 h-1, when [Ce(IV)]0 = [glucamine]0 = 10 mmol dm-3. The DNA-hydrolyzing activity decreases in the following order: glucamine > isomaltose, melibiose, gentiobiose >> palatinose, mannitol, sorbitol, galactitol.

Acetylacetonato-lanthanide complexes as eminent catalytic sites for artificial ribonucleases.

Acetylacetonato complexes of lutetium, ytterbium, thulium, and europium ions efficiently hydrolyze the phosphodiester linkage in adenylyl(3'-5')adenosine. The pseudo first-order rate constant (3.2 x 10(-2) h-1) at pH 7.2 and 30 degrees C for the 1:1 lutetium-acetylacetonato complex (0.5 mmol dm-3) is close to the value (3.5 x 10(-2) h-1) for free lutetium ion of the same concentration. Potentialities of these complexes as the catalytic sites of artificial ribonucleases are indicated.

Hydrolysis of DNA by Ce(IV)/EDTA complexes--the mechanism of DNA cleavage and design of artificial restriction enzymes.

Homogeneous Ce(IV) complex of EDTA promptly hydrolyzes oligonucleotides under physiological conditions. Moreover, the activity of Ce(IV)/EDTA for DNA hydrolysis is promoted by the addition of amines. When [Ce(IV)/EDTA] = 5 mumol dm3 and [ethylenediamine] = 100 mmol dm3, the catalytic activity is about 50 times as large as that of Ce(IV)/EDTA. The combination of Ce(IV)/EDTA and amines is eminent tools for the future molecular biology and biotechnology.

Trinuclear Cu(II) complex for internucleoside linkage-specific cleavage of RNA.

Trinuclear Cu(II) complex of N,N,N',N',N",N"-hexa[(2-pyridyl)methyl]-1,3,5-tris(aminomethyl)benzene (L3A) exhibits remarkable substrate-specificity for hydrolysis of 2'-->5' and 3'-->5' ribonucleotides. Up(2'-->5')U is hydrolyzed much efficiently than is Up(3'-->5')U, but Ap(3'-->5')A is overwhelmingly preferable to Ap(2'-->5')A.