Bonds cleaving enzymes

The Catalytic Strategy of P-0 Bond-Cleaving Enzymes Comparing EcoRV and Myosin... 

The sequences are written 5 —3 in the top strand and 3 —5 in the bottom strand. The phosphodiester bonds cleaved by each enzyme are marked T in the top strand and T in the bottom strand. The shorthand used to identify specific restriction enzymes can be confusing to a student reading a scientific paper, in which the shorthand is not explained. 

Any electromechanical device that utilizes an automated feedback servomotor to regulate the addition of titrant (a standardized solution of acid or base within a syringe) into a reaction vessel or sample to maintain pH. The rate at which the syringe expels its contents allows one to determine the rate of a chemical reaction producing or consuming protons. There are many such enzyme-catalyzed reactions whose kinetics can be examined with a pH Stat. For maximal sensitivity, one must use weakly buffered solutions. In his classical kinetic investigation of DNA bond scission by DNase, Thomas measured the rate of base addition in a pH Stat. The number of bonds cleaved was linear with time, and this was indicative of random scission. 

Adjacent half-cystine residues are present in many peptides and proteins. In most of the cases they form two disulfide bonds with other cysteines in the molecule, unless the peptide bond between them is cis (see Section 6.1.5.1 ).t40 41 Specific enzymes that cleave the Cys-Xaa bond have not yet been discovered, although there are a few reports of cleavage of the Cys-Cys bond by enzymes such as elastase and pepsinJ42-43 For peptides with Cys-Cys bonds the cleavage method in Section 6.1.6.2.4 is recommended. 

Phospholipases are enzymes that cleave phospholipids. Phosphohpases of type Al, A2, C and D are differentiated according to the specificity of the attack point on the phospholipid. The bonds cleaved by these phospholipases are shown in Fig. 5.24a. 

Arrows indicate the phosphodiester bonds cleaved by each restriction endonuclease, Asterisks indicate bases that are methylated by the corresponding methylase (where known), N denotes any base. Note that the name of each enzyme consists of a three-letter abbreviation (in italics) of the bacterial species from which it is derived, sometimes followed by a strain designation and Roman numerals to distinguish different restriction endonucleases isolated from the same bacterial species, Thus BamHI is the first (I) restriction endonuclease characterized from Bacillus amyloliquefaciens, strain H. 

Enzyme Pathway(s) Bond cleaved Bond formed... 

In Fig. 14-5 the reactions of PLP-amino acid Schiff bases are compared with those of (i-oxo-acids. Beta-hydroxy-a-oxo acids and Schiff bases of PLP with (i-hydroxy-a-amino acids can react in similar ways. The reactions fall naturally into three groups (a,b,c) depending upon whether the bond cleaved is from the a-carbon of the substrate to the hydrogen atom, to the carboxyl group, or to the side chain. A fourth group of reactions of PLP-dependent enzymes (d) also involve removal of the a-hydrogen but are mechanistically more complex. Some of the many reactions catalyzed by these enzymes are listed in Table 14-3. 

In any quantitative work on protein hydrolysis, it is necessary to have a measure of the extent of the hydrolytic degradation. The measurement of the number of peptide bonds cleaved during a hydrolytic process is related to the activity of proteinolytic enzymes and the extent of hydrolysis. Various techniques that evaluate the progress of hydrolysis have been reported, such as the trichloroacetic acid (TCA) solubility index, which evaluates the percentage of nitrogen soluble in TCA after partial hydrolysis of the protein. 

Ribonuclease IV purified from an RNase I minus strain of E. coli (MRE-600) specifically cleaves R17 RNA into two large fragments, one sedimenting at about 15 S carrying the 5 terminal of the original molecule, and the other sedimenting at about 21 S (131). The nature of the bonds cleaved by the enzyme is not known. 

The role of the nucleoside triphosphate in the hydrolysis of DNA has not yet been clarified. ATP and dATP are the most effective nucleotides and only slight activity (10% or less) is observed with the other triphosphates nucleoside diphosphates are inactive. The rate of DNA hydrolysis is proportional to the ATP concentration and the ATP is converted to ADP and inorganic phosphate in the course of the reaction. Three moles of ATP are consumed for each phosphodiester bond cleaved, indicating a complex mechanism of participation of ATP in the endonucleolytic reaction. Preliminary experiments by Takagi and his colleagues indicate that the purified enzyme catalyzes an exchange of ADP with ATP in the absence of DNA, suggesting that a phospho enzyme may be an intermediate. 

Because the transaldolase and transketolase reactions are symmetrical with respect to types of bonds cleaved and formed, their equilibrium constants are near 1. The pool of sugar phosphates is thus near equilibrium in cells that contain these enzymes. In a water-flow analogy, the sugar phosphates and the reactions that interconvert them resemble a large swamp, with ill-defined flows along many interconnecting channels. Water may be fed in from any direction and may leave the swamp in any direction. 

Enzymes that degrade phospholipids are called phospholipases. They are classified according to the bond cleaved in a phospholipid (fig. 19.11). Phospholipases Al and A2 selec- tively remove fatty acids from the sn-1 and sn-2 positions, i respectively. Phospholipase C cleaves between glycerol and the phosphate moieties phospholipase D hydrolyzes the [ head-group moiety X from the phospholipid. Lysophospho- lipids, which lack a fatty acid at the sn-1 or sn-2 position, j are degraded by lysophospholipases. f Phospholipases are found in all types of cells and in... 

The proton donated from the OH group of Ser 195 to His 57 is then donated to the N atom of the scissile bond, cleaving the C-N peptide bond (or the C-O ester bond) to produce the amine and the acyl-enzyme intermediate. The amine is that part of the substrate which follows the scissile bond in the sequence the acyl-enzyme intermediate is the remaining fragment covalently bound to Ser 195. 

Figure 2. A representation of the interaction of a polypeptide substrate with the extended substrate binding site of a protease. The individual amino acid (AA) residues are designated Pl7 P2, P/ etc. and the corresponding subsites of the enzyme are Sl7 S2, S/ etc. The bond cleaved by the enzyme is the peptide bond between the Pt and P/ residues.

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