Aminotransferases modified

Glutamate Glutamate dehydrogenase/ alanine aminotransferase Modified carbon electrode... 

In bi-substrate reactions of the double displacement type, one substrate must be bound and one product released before the entry of the second substrate. In such reactions, the first substrate reacts with the enzyme to yield a chemically modified form of the enzyme (usually a functional group is changed) and the first product. In the second step, the functional group of the modified enzyme is transferred from the enzyme to the second substrate to form the second product. A good example is the aminotransferase class of enzymes, where an amino group is transferred from an amino-acid to the enzyme, from which it is transfered to a keto-acid. 

In double-displacement, or Ping-Pong, reactions, one or more products are released before all substrates bind the enzyme. The defining feature of double-displacement reactions is the existence of a substituted enzyme intermediate, in which the enzyme is temporarily modified. Reactions that shuttle amino groups between amino acids and a-keto acids are classic examples of double-displacement mechanisms. The enzyme aspartate aminotransferase (Section 23.3.1) catalyzes the transfer of an amino group from aspartate to a-ketoglutarate. 

Figure 21-3 The ratio (mean SD) of mitochondrial to total AST in hepatic diseases.The difference between alcoholic hepatitis and other hepatic diseases is statistically significant (p < 0.05). (Modified from Panteghini A1, FalseW f Chiari , MalchiodiA, Determination of aspartate aminotransferase isoenzymes in hepatic diseases. Lab j Res Lab Med 1983 i 0 515-9.)...

In a double-displacement reaction, at first only one substrate is bound the release of one product and covalently modified enzyme (E ) follows. E then combines with the second substrate to form a second product. Reactions catalyzed by aminotransferases are of this type. In this mechanism, no ternary complex EAB is formed. The double displacement reaction sequence is shown below ... 

Fig. 7. Glutaminase, mitochondrial and soluble aspartate aminotransferases and glutamate dehydrogenase im-munoreactivities in the cerebral neocortex of the rat. The figure is modified from Kaneko and Mizuno (1994). PAG = phosphate-activated glutaminase.

In a recycling system using glutamate dehydrogenase and alanine aminotransferase the direct oxidation of the liberated NADH at an electrode modified with Meldola s Blue as well as the NADH oxidation by NMP+ and the subsequent detection of the oxygen consumption by... 

ADP AFP ab as ALAT AP ASAT ATP BQ BSA CEH CK CME COD con A CV d D E E EC ECME EDTA EIA /e FAD FET FIA G GOD G6P-DH HBg HCG adenosine diphosphate a-fetoprotein antibody antigen alanine aminotranferase alkaline phosphatase aspartate aminotransferase adenosine triphosphate benzoquinone bovine serum albumin cholesterol ester hydrolase creatine kinase chemically modified electrode cholesterol oxidase concanavalin A coefficient of variation (relative standard deviation) layer thickness diffusion coefficient enzyme potential Enzyme Classification enzyme-chemically modified electrode ethylene diamine tetraacetic acid enzyme immunoassay enzyme loading factor flavin adenine dinucleotide field effect transistor flow injection analysis amplification factor glucose oxidase glucose-6-phosphate dehydrogenase hepatitis B surface antigen human chorionic gonadotropin... 

In an attempt to reverse the substrate charge specificity for aspartate aminotransferase, an Arg-292 residue which forms a salt bridge with the carboxylate on the side chain of the substrate aspartate was modified to an Asp (60). Model building studies based on the crystal structure of the wild-type enzyme indicated that the Arg-292+ Asp ion pair could be replaced by an Asp-292 + Arg" pair in the mutant (Fig. 7). The specificity of the Arg-292 -> Asp mutant in terms of... 

Confirmation of the molecular structure of the enzyme-inactivator adduct has been obtained for few modified PLP-dependent enzymes. In the case of the reaction of aspartate transaminase (aspartate aminotransferase) with L-serine 0-sulfate, the surprising result thus obtained by Metzler and co-workers has forced reevaluation of the mechanism of similar inactivators (Ueno et al., 1982). Conventional wisdom argued that the reaction should involve elimination of sulfate from the inactivator followed by addition of an enzyme nucleophile to the resulting double bond (Fig. 8). When subjected to high pH, however, the inactivated enzyme releases a yellow PLP adduct which has been identified as the aldol product of the cofactor and C-3 of pyruvate (9, Fig. 9) as previously prepared by... 

The pattern recorded above raises the question whether the change of face at C-4 to the solvent side is a mandatory requirement in the transformation of binary into ternary complexes in pyridoxal-P-dependent reactions. That this may be so was the view beginning to prevail until a timely reminder, or perhaps an undue caution, came from a more recent report by Zito and Martinez-Carrion [93]. As has already been cited, these workers repeated the earlier experiments of the Zurich School on aspartate aminotransferase confirming the Re face hydride attack at C-4 in the binary complex. However, aspartate aminotransferase carbamylated at the active site Lys-258 was used to produce the substrate-coenzyme Schiff base linkage in the ternary complex. Since the modified enzyme catalysed the half-transamination reaction ... 

Besides this main effect, which leads to suffocation, carbon monoxide also exerts other effects. They are, however, of a lower importance in a comparison with the effect on haemoglobin. It is possible to consider effects on myoglobin and on the other tetrapyrol substances. Many enzymes containing trace elements modify their activity by the action of carbon monoxide [41]. An increase of the activity of aspartate-aminotransferase and of the lactate concentration can be used for the indication of acute poisoning [42]. 

See color insert.) Subject liver function tests by trial day at-risk subjects. For alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (Aik Phos), the clinical concern level (CCL) is two times upper limit of normal (ULN) for total bilirubin, the CCL is 1.5x ULN. Shaded region indicates time on treatment. (From CTSpedia, http //www.ctspedia.org/do/view/CTSpedia/ClinLFTGraph005, accessed January 8, 2013. Code by Susan Schwartz, modified by Robert Gordon.)... 

The potential of biotransformations with genetically modified microorganisms can be illustrated by the following example, where the chain of added-value to amino acid products is also recognisable The Mercian Company (Japan) uses a recombinant E. coli strain to prepare (S)-piperidine-2-car-boxylic acid from (L)-lysine. In this strain, the (L)-lysine-permease transport system is overexpressed, so that in this pathway lysine is produced efficiently in the cells. The bacteria possess additionally a (L)-lysine-aminotransferase from Flavobacterium lutescens, which brings about the deamination of lysine. The thus generated aldehyde is in equilibrium with its intramolecular imine, which, in presence of the E. co/i-specific pyrroline-5-carboxylate reductase, is reduced with NADPH to (S)-piperidine-2-carboxylic acid. The turnover... 

A range of (D)-amino acids is also accessible by a fermentation route. The microorganisms are for this purpose modified, so that they no longer express functional (D)-amino acid deaminases. However, they contain instead genes for (L)-amino acid deaminases, (D)-amino acid aminotransferases and a special racemase. Monsanto, for example, has patented such an approach for the preparation of (D)-phenylalanine. 

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