P-O bond-cleaving enzymes

I 77 The Catalytic Strategy of P-O Bond-Cleaving Enzymes Comparing EcoRV and Myosin... 

The use of isolated enzymes to form or cleave P-O bonds is an important application of biocatalysts. Restriction endonucleases, (deoxy)ribonucleases, DNA/ RNA-ligases, DNA-RNA-polymerases, reverse transcriptases etc. are central to modern molecular biology(1). Enzyme catalyzed phosphoryl transfer reactions have also found important applications in synthetic organic chemistry. In particular, the development of convenient cofactor regeneration systems has made possible the practical scale synthesis of carbohydrates, nucleoside phosphates, nucleoside phosphate sugars and other natural products and their analogs. This chapter gives an overview of this field of research. 

EcoRV is a hydrolase enzyme that belongs to the nuclease enzymes (EC 3.1) and is responsible for cleaving the P-O bond of phosphodiester in DNA and RNA. Exonuclease enzymes hydrolyze the P-O phosphodiester bond of the terminal mononucleotide and are not sequence specific [31]. Endonuclease enzymes include both examples, that is, enzymes that are not sequence specific and those that are sequence specific (e.g., type II restriction endonucleases) [32]. Type II restriction endonucleases in bacteria, archaea, and algae use their sequence specificity to bind and cleave the foreign DNA of invading viruses. These enzymes are especially interesting, as they combine their remarkable sequence specificity with high catalytic rates of phosphodiester hydrolysis. 

Ribonucleases are a widely distributed family of en-zymes that hydrolyze RNA by cutting the P—O ester bond attached to a ribose 5 carbon (fig. 8.12). A good representative of the family is the pancreatic enzyme ribonuclease A (RNase A), which is specific for a pyrimidine base (uracil or cytosine) on the 3 side of the phosphate bond that is cleaved. When the amino acid sequence of bovine RNase A was determined in 1960 by Stanford Moore and William Stein, it was the first enzyme and only the second protein to be sequenced. RNase A thus played an important role in the development of ideas about enzymatic catalysis. It was one of the first enzymes to have its three-dimensional structure elucidated by x-ray diffraction and was also the first to be synthesized completely from its amino acids. The synthetic protein proved to be enzymatically indistinguishable from the native enzyme. 

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). 

Akhtar, M., VC.O. Njar, and J.N. Wright (1993). Mechanistic studies on aromatase and related carbon-carbon bond cleaving P-450 enzymes. J. 

The enzymatic hydrolysis of lecithin to fatty acids, glycerol, phosphoric acid, and choline requires the participation of some four enzymes. The enzymes attacking the various bonds of a lecithin molecule are summarized in Table XIV. The bonds which are cleaved are referred to by number in the lecithin formula shown below the same numbers are used in Table XIV to indicate the respective enzymes involved. The cleavage of the fatty acid-glycerol bond, as represented by diagonal lines 1 and 2, is based on hydrolysis studies of simple esters. Diagonal lines 3 and 4 are only provisional, as presumably either a P—0 or C—O bond to phosphate could be cleaved. 

By proton inventory, a technique that determines whether acid and base groups act simultaneously, we found that hydrolysis of 36 by artificial enzyme 44 involves two protons moving in the transition state [130]. Thus, ImH+ of 46 is hydrogen bonded to a phosphate oxyanion of bound substrate 36 water hydrogen bonded to the Im then attacks the phosphorus, and as the O-P bond forms the ImH+ proton transfers (along with the water proton) to produce the phosphorane monoanion 47. This then goes on to the cleaved product in later catalyzed steps before there is time for pseudo-rotation. These general conclusions have been described and summarized in several publications [131-137]. 

DNase I is an endonuclease that catalyzes the hydrolysis of phosphodiester bonds by nucleophilic attack on 3 O-P. The enzyme activity requires divalent metal ion cofactors such as Mg " and Ca or Mn +. DNase I binds to the minor groove of dsDNA and cleaves each strand independently. The often observed sequence preference and different cleavage rates of DNase I are largely structural and are related to sequence-dependent variations of the double helix such as groove width, local rigidity to bending, radial asymmetry, and accessibility to backbone phosphates (26,29,33,34). 

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