Amino acid pyrophosphatase

From Sigma 3-aminoethylcarbazole (AEC) acrylamide/bis-acrylamide (30%) 37.5 1 amino acids alumina bentonite benzamidine bovine fiver tRNA bovine serum albumin (BSA) creatine phosphate (CP) diethyl pyrocarbonate (DEPC) dithiothreitol (DTT) Escherichia coli MRE600 tRNA pyrophosphatase (Ppase) Ca++ salt of folinic acid, (5-formyl THF) IIHPHS K salt of phospho-enol pyruvic acid, (PEP) creatine phospho kinase (CPK) protease inhibitor cocktail for fungal and yeast extracts phenylmethylsulfonyl fluoride (PMSF) spermidine trihydrochloride Tween 20. 

In 1998 the long-known Bacillus subtilis inorganic pyrophosphatase was characterized and found to have much greater activity than the above enzymes, to have a completely different amino acid sequence, not to be inhibited by F and to be activated by This form of... 

This overall reaction is reversible but is driven to completion by the subsequent hydrolysis of PP, to two equivalents of P through the action of ubiquitous pyrophosphatases. Thus, the formation of aminoacyl-tRNA consumes two equivalents of ATP. The energy that ultimately drives the formation of the peptide linkage during protein synthesis is derived from the ester linkage that joins amino acids to tRNA. 

Aminoacylation is a two-step process, catalyzed by a set of enzymes known as aminoacyl-tRNA synthetases. Twenty aminoacyl-tRNA synthetases reside in each cell, one per amino acid in the genetic code. In the first step of aminoacyl-tRNA synthesis, ATP and the appropriate amino acid form an aminoacyl adenylate intermediate. Inorganic pyrophosphate is released and subsequently broken down to free phosphate by the enzyme inorganic pyrophosphatase. The aminoacyl adenylate is a high-energy intermediate, and in the second step, the transfer of amino acids to the acceptor end of tRNA occurs without any further input of ATP, as shown in Figure 11-2. 

The pyrophosphate thus produced is often degraded by a ubiquitous enzyme, pyrophosphatase, to inorganic phosphate, thus pushing the reaction ever further toward completion. Another process that requires energy input is the formation of peptide bonds, which hold amino acids together in proteins. Peptide bonds cannot form spontaneously, and their biosynthesis requires at least four ATP equivalents, amounting to some 33,600 cal. The whole point of ATP hydrolysis in biologic systems is thus to drive forward those reactions that would otherwise not occur because of unfavorable AG. ... 

The splitting of inorganic pyrophosphafe (PPj) into two inorganic phosphate ions is catalyzed by pyrophosphatases (p. 636) that apparently occur universally. Their function appears to be simply to remove the product PPj from reactions that produce it, shifting the equilibrium toward formation of a desired compound. An example is the formation of aminoacyl-tRNA molecules needed for protein synthesis. As shown in Eq. 17-36, the process requires the use of two ATP molecules to activate one amino acid. While the "spending" of two ATPs for the addition of one monomer imif to a polymer does not appear necessary from a thermodynamic viewpoint, it is frequently observed, and there is no doubt that hydrolysis of PP ensures thaf the reaction will go virtually to completion. Transfer RNAs fend to become saturated with amino acids according to Eq. 17-36 even if the concentration of free amino acid in the cytoplasm is low. On the other hand, kinetic considerations may be involved. Perhaps the biosynthetic sequence would move too slowly if if were nof for the extra boost given by the removal of PPj. Part of the explanation for the complexity may depend on control mechanisms which are only incompletely understood. 

FIGURE 12.6 The aminoacyl-tRNA synthetase reaction, (a) The overall reaction. Everpresent pyrophosphatases in cells quickly hydrolyze the PPj produced in the aminoacyl-tRNA synthetase reaction, rendering aminoacyl-tRNA synthesis thermodynamically favorable and essentially irreversible, (b) The overall reaction commonly proceeds in two steps (i) formation of an aminoacyl-adenylate and (ii) transfer of the activated amino acid moiety of the mixed anhydride to either the 2 -OH (class I aminoacyl-tRNA synthetases) or 3 -OH (class II aminoacyl-tRNA synthetases) of the ribose on the terminal adenylic acid at the 3 -OH terminus common to all tRNAs. Those aminoacyl-tRNAs formed as 2 -OH esters undergo a transesterification that moves the aminoacyl group to the 3 -OH of tRNA Only the 3 -esters are substrates for protein synthesis. 

In subsequent studies (Kenney et ai, 1974a, 1976a) of the flavin prostjietic group of the j -cyclopiazonate oxidocyclases, the isoenzymes were digested with trypsin and chymotrypsin. A flavin mononucleotide peptide was isolated from the resultant mixture by chromatography on Florisil and diethylaminoethyl cellulose and by hydrolysis with nucleotide pyrophosphatase. Its amino acid composition was determined after hydrolysis in... 

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