Specificity and cofactors

The enzymatic processes involved in the formation of catecholamines have been characterized. The component enzymes in the pathway have been purified to homogeneity, which has allowed for detailed analysis of their kinetics, substrate specificity and cofactor requirements and forthe development of inhibitors (Fig. 12-l).TheircDNAs have been cloned, and studies with knockout mice clearly indicate the importance of these enzymes since their... 

Like LAR, ANR is a member of the RED protein family. Full details of the protein structure and reaction mechanism are yet to be published. However, Xie et al. compared the A. thaliana (AtANR) and M. truncatula (MtANR) ANR amino acid sequences and recombinant protein activities, and made suggestions on the possible reaction series. The two recombinant proteins showed significantly different kinetic properties, substrate specificities, and cofactor requirements. Although AtANR and MtANR share only 60% sequence identity, some well-conserved domains are evident, in particular the Rossmann dinucleotidebinding domain (GxxGxxG) near the N-termini. However, two amino acid variations did... 

Reed J. R., Hernandez R, Blomquist G. J., Feyereisen R. and Reitz R. C. (1996) Hydrocarbon biosynthesis in the housefly, Musca domestica substrate specificity and cofactor requirement of P450hyd. Insect Biochem. Molec. Biol. 26, 267-276. 

When the appropriate enzyme reaction was detected, a rough enzyme characterization was performed including determination of the molecular mass, pH optimum, isoelectric point, preliniinary substrate acceptance (substrate specificity), and cofactor studies. 

A further way in which metabolic control may be exercised is the artificial deprivation of required ions and cofactors, for example aconitase must have ferrous ions for activity. Conversely, addition of toxic ions is possible, for example aconitase is inhibited by cupric ions. Finally the use of metabolic analogues is possible. If monofluoroacetate is added to cells then monofluorocitrate is produced by titrate synthase and this compound inhibits the activity of aconitase. Great care has to be taken when using metabolic analogues, however, they are often less than 100% specific and may have unexpected and unwanted serious side effects. 

Lipke H, CW Kearns (1959b) DDT dehydrochlorinase II. Substrate and cofactor specificity. J Biol Chem 234 ... 

Dioxygenases often have broad substrate specificity and require only a minimal characteristic structure for substrate recognition [310], Transition metal or an organic cofactor mediates dioxygen activation needed by the oxygenases action. Iron and copper, in their lower oxidation states are the metals most commonly used, but also organic co-factors like dihydroflavin and tetrahydropterin are able to activate the oxygen molecule. 

The fact that a receptor dimer identifies a HRE does not assure, by itself, the transcription of the gene. This is a necessary, but insufficient, condition. Once the dimer-HRE interaction has been produced, the machinery of transcription needs to be assembled, requiring the binding of other intermediary cofactors. Some of these are tissue specific, and others recognize only a particular receptor dimer, thus obviating others that could recognize the same HRE. 

This enzyme [EC 2.6.1.1] (also known as transaminase A, glutamicioxaloacetic transaminase, and glutamic aspartic transaminase) catalyzes the reversible reaction of aspartate with a-ketoglutarate to produce oxaloace-tate and glutamate. Pyridoxal phosphate is a required cofactor. The enzyme has a relatively broad specificity, and tyrosine, phenylalanine, and tryptophan can all serve as substrates. 

Current available information does not permit definitive conclusions on the nature, specificity, and mechanism of action of the protein cofactor (s) of lipoprotein lipase. It is verj difiicult to correlate the observations described above (summarized in Table 10) since the enzyme preparations used were not pure or well characterized, and were derived from various sources. For instance, two species of lipoprotein lipase have been reported to exist in rat adipose tissue (G4), and major differences between enzymes of liver and adipose tissue have been noted (G16). Also, the nature of the apoprotein preparations employed as protein cofactor (s) of lipoprotein lipase has not been clearly specified in all the studies contaminated materials may account for the spurious results observed. At present, it is not known how apoproteins such as apo Glu, apo Ala, and apo Ser could exhibit their activator or inhibitor activity on lipoprotein lipase. If these different apoproteins indeed prove to be cofactors for lipoprotein lipase, the nature of the lipid-protein specificity must be established and thus the role played by carbohydrates, since some of these apoproteins are glycoproteins. 

However, this glycine-rich segment has other functions in short-chain alcohol dehydrogenases. Tanaka et al. [37] and our 3D modeling [60] indicate that this glycine rich segment has an important role in cofactor specificity and binding of the nicotinamide moiety to 11P-HSD. 

The structure of the ALR2 holoenzyme showed that the catalytic site was situated atop the nicotinamide moiety of the NADPH cofactor. The substrate binding site, which would determine the enzyme s specificity and also presumably bind inhibitors, appeared to be composed of a deep cleft (Figures 3 and 4). It extended away from the catalytic site towards the loop composed of residues between (34 and cc4 and the last 20 residues of the carboxy-terminal meander. This hypothesis was supported by the appearance of poorly resolved density that occupied this region, which suggested the presence of an endogenously bound substrate or inhibitor in the structure of the holoenzyme [16]. Subsequent studies indicate that this electron density may be a citrate molecule, one of the components included in the crystallization mixture. Activity studies indicate that citrate is indeed one of the many inhibitors of the enzyme with a K in the millimolar range [23]. 

Folding and posttranslational Specific enzymes, cofactors, and other components for... 

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