Substrates Tyrosine

Tyrosine kinase inhibitors interfere with the function of tyrosine kinases that catalyze the transfer of the y-phosphate group of ATP to tyrosine residues of protein substrates. Tyrosine kinases can be subdivided into two large families, receptor tyrosine kinases (RTKs) and non-receptor tyrosine kinases (NRTKs see corresponding chapter). The human genome... 

Hydrogen bonding possibly occurs between the substrate tyrosine amide unshared pair and the side-chain HO groups of the enzyme tyrosine 248 (Y). 

In the case of oligomeric proteins in which subunit contact regions have been revealed by X-ray crystallography34 353 or other methods described above,363 the equilibrium between oligomer and monomer can be changed by site-directed mutagenesis. For example, stable monomers of tyrosyl-tRNA synthetase were produced by a mutation of Phe-164 at the subunit interface to Asp, and it was revealed that the monomers are inactive and do not bind the substrate tyrosine.343 In the case of yeast triosephosphate isomerase, replacement of Asn-78 at the subunit interface did not cause dissociation of subunits under normal conditions.353 However, the stability of the enzyme was significantly lowered by the mutation, probably due to decreased subunit-subunit interaction.353... 

A third amino acid hydroxylase, tyrosine hydroxylase, causes the NIH Shift with phenylalanine (21) but not with its normal substrate, tyrosine (Figure 3). Thus, the hydroxylation of 4-tritiophenylalanine with either tyrosine hydroxylase or phenylalanine hydroxylase leads to migration and retention of tritium in the product, tyrosine. However, with 3,5-ditritiotyrosine as substrate for tyrosine hydroxylase, complete loss of one tritium occurs in the conversion to 3,4-dihydroxy-5-tritio-phenylalanine. 

The conventional substrates, tyrosine and dopa, can stimulate tyrosinase activity as well as melanization of the cells (766, 206, 248, 249). Positive regulation of tyrosinase by its precursors in vivo that require active protein synthesis was shown for the first time by Slominski et al. (246, 247). Both L-tyrosinase and L-dopa stimulate tyrosinase at the level of translation (248). Depending on dose and time, L-dopa can both stimulate and inhibit tyrosinase mRNA expression (249). Recently, it has been found that tryptophan can stimulate new mRNA-dependent tyrosinase synthesis in B16 murine melanoma cells (38). 

Since it was necessary to use ethanol to keep quercetin in solution, we tested effects of ethanol on tyrosinase activity with its usual substrate, tyrosine (Worthington, 1982). The following results were obtained (in units/mg) pH 6.5 Tris buffer, 3,000 50% pH 6.5 buffer-50% ethanol, 700 pH 7.0 buffer, 2,500 50% pH 7.0 buffer-50% ethanol, 1,000 pH 7.5 buffer, 2,650 50% pH 7.5 buffer-50% ethanol, 1,100. Although ethanol suppresses tyrosinase activity, it does not completely inactivate the enzyme under conditions of the present study. Additional studies revealed that the extent of suppression is directly related to the amount of ethanol in the mixed solvent. 

The enzymatic method described above has two disadvantages (1) trapping of CO2 is a cumbersome procedure, and (2) the use of a radioactive substrate requires special precautions for use and disposal of reagents. Measurement of the primary amine formed by decarboxylation of the amino acid can also be exploited to monitor the PLP-dependent, enzyme-catalyzed reaction. This principle has been applied by Allenmark et al. (106), who used L-3,4-dihydroxyphenyl-alanine (L-DOPA) as substrate for tyrosine decarboxylase the dopamine produced by the decarboxylation reaction was determined by HPLC followed by amperometric detection. Both Hamfelt (107) and Lequeu et al. (108) utilized apo-tyrosine decarboxylase with tyrosine as substrate. The tyramine produced by the decarboxylation reaction was separated from the substrate (tyrosine) by HPLC and quantitated by either amperometric (108) or fluorometric (107) detection. The procedures discussed above are still subject to the main disadvantage of enzymatic methods possible interference by other materials present in the PLP containing extract which could either inhibit reconstitution of the holoenzyme or alter the reaction rate of enzyme catalysis. Moreover, HPLC with amperometric detection can hardly be described as less cumbersome than CO2 trapping difficulties in baseline-stabilization encountered with these detectors are well known. 

PAL enzyme has been found to be widely distributed whereas the enzyme performing an analagous reaction with tyrosine as substrate, tyrosine ammonia lyase (TAL), has so far only been detected in grasses. 

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