Catalysis nuclease

En me Mechanism. Staphylococcal nuclease (SNase) accelerates the hydrolysis of phosphodiester bonds in nucleic acids (qv) some 10 -fold over the uncatalyzed rate (r93 and references therein). Mutagenesis studies in which Glu43 has been replaced by Asp or Gin have shown Glu to be important for high catalytic activity. The enzyme mechanism is thought to involve base catalysis in which Glu43 acts as a general base and activates a water molecule that attacks the phosphodiester backbone of DNA. To study this mechanistic possibiUty further, Glu was replaced by two unnatural amino acids. 

Yet another super family, the nucleotidyl-transferase family, also utilizes two-metal-ion-dependent catalysis the members include transposases, retrovirus integrases and Holliday junction resolvases4. Whereas in the nucleases, the Mg2+ ions are asymmetrically coordinated, and play distinct roles, in activating the nucleophile and stabilizing the transition state, respectively, in the transposases, they are symmetrically coordinated and exchange roles to alternatively activate a water molecule and a 3 -OH for successive strand cleavage and transfer. 

Staphylococcal nuclease is a phosphodiesterase which can cleave either DNA or RNA to produce 3 -phosphomononucleosides (3-16, 20,25). The rates of hydrolysis of these substrates are dependent on the conformation of the substrate, Ca2+ concentration, and the ionic strength and the pH of the buffer (3, 54). Denatured DNA is hydrolzed more rapidly than native DNA (3, 12, 54-56), which reflects the important effect of substrate conformation on catalysis. In native DNA the Xp-dTp and Xp-dAp bonds are preferentially attached (7, 12, 14, 15, 55). With denatured DNA the order of cleavage appears to be nearly random (14, 15, 56, 57). The Xp-dCp and Xp-dGp linkages in the helical regions of DNA, which are more extensively stabilized, are more resistant to hydrolysis. The specific order of release of various mononucleotides from native compared to denatured DNA suggests that in the hydrolysis of DNA specificity toward the constitutent bases is less important than the substrate conformation (54-57). 

Interactions of the same water molecules with RNA nucleotides (via H-bonding) and metal ions (via inner-sphere coordination) could stabilize specific metal ion-nucleic acid complexes (e.g. in Mg + -tRNA chelates) and also create the possibility for direct proton transfer through a water chain that could play a role in ribozyme-metal ion catalysis and in the mechanism of metal-dependent nucleases and polymerases. Similar types of H-bonds between different nucleotide residues have been found in tRNA tertiary structures, where they provide additional stabilization of tertiary interactions. 

Figure 15. Reaction mechanism for staphylococcal nuclease, (a) gcncral-basc catalysis, (b) nucleophilic attacl by a hydroxyl anion, (c) cleavage of the 5 0-P bond.

Staphylococcal nuclease (SNase) is one of the most powerful enzymes known in terms of its rate acceleration, with a catalytic rate that exceeds that of the non-enzymatic reaction by as much as 1016.211 This enzyme is a phosphodiesterase, and utilizes a Ca2+ ion for catalysis to hydrolyze the linkages in DNA and RNA. In addition to the metal ion, the active site has two Arg residues in a position to interact with the phosphoryl group, and a glutamate. X-ray structures212 215 of SNase have been solved for the wild-type enzyme and mutants, but the exact roles of active-site residues are still uncertain. SNase cleaves the 5 O-P nucleotide bond to yield a free 5 -hydroxyl group (Fig. 30). 

Weiss and coworkers pioneered in using single-molecule FRET to study the conformational dynamics and reaction mechanism of staphylococcal nuclease.85 Lu and coworkers used it to study the conformational dynamics of T4 lysozyme during catalysis.58 Hammes, Benkovic, and coworkers studied dihydrofolate reductase,25 the enzymes involved in T4 primosome86 and replisome.87 88 Yang and coworkers studied adenylate kinase.89 Here, we use the T4 lysozyme study to illustrate the approach.5... 

This chapter focuses on reactivity control in stoichiometric and catalytic reactions taking place in the confines of supramolecnlar complexes of reactants with calixarene receptors. Earlier work on the subject was reviewed by us in 2000. Quite recently, Homden and Redshaw have published an extensive review on the use of calixarenes in metal-based catalysis. Because of space limitations and, even more importantly, to avoid extensive overlap with the already reviewed material, the scope of this chapter is restricted to works treated only marginally, or not treated at aU, in the above review article. The first section deals with examples in which reactivity control takes place via substrate inclusion into the calixarene cavity. The other section illustrates the use of the calix[4] aiene upper rim in the construction of di- and trimetalfic complexes capable of esterase and nuclease activity. 

---