Phenylalanine inhibition

In the case of hyperphenylalaninaemia, which occurs ia phenylketonuria because of a congenital absence of phenylalanine hydroxylase, the observed phenylalanine inhibition of proteia synthesis may result from competition between T.-phenylalanine and L-methionine for methionyl-/RNA. Patients sufferiag from maple symp urine disease, an inborn lack of branched chain oxo acid decarboxylase, are mentally retarded unless the condition is treated early enough. It is possible that the high level of branched-chain amino acids inhibits uptake of L-tryptophan and L-tyrosiae iato the brain. Brain iajury of mice within ten days after thek bkth was reported as a result of hypodermic kijections of monosodium glutamate (MSG) (0.5—4 g/kg). However, the FDA concluded that MSG is a safe kigredient, because mice are bom with underdeveloped brains regardless of MSG kijections (106). 

An alkaline pH (- pH 11) is desirable in order to achieve high conversion rates increase solubility of L-phenylalanine inhibit enzymes catalysing degradation of L-phenylalanine and formation of byproducts reduce inhibition of the reaction by the keto form of phenylpyruvic arid. 

It was shown <1999BML3255> that a crystalline ozonide obtained by ozonolysis of the A-allylamidc of Cbz-L-phenylalanine inhibits papain, a cysteine protease. Reduction of that ozonide in excess dimethyl sulfoxide (DMSO) generates in situ a peptide aldehyde, as proved by coupling with a stabilized Wittig ylide forming thereby an unsaturated ester. 

The LAT system has been used for the transport of various compounds to the brain. Variations in the cerebellum to plasma ratio at late times in 6-[18F]fluoro-L-DOPA studies are consistent with competitive binding of large neutral amino acids (LNAAs) for the LAT at the BBB (117). In addition, it was shown that oral administration of phenylalanine inhibited the uptake of an artificial amino acid [(1 lC)-aminocyclohexanecarboxylate] in human brain (118). Melphalan, a nitrogen mustard derivative of the neutral amino acid L-phenylalanine, was transported to the brain via the LAT system at the rat BBB. In addition, it was shown that melphalan competed with phenylalanine for the LAT system (119). 

An attempt was made to find conditions in which human intestinal alkaline phosphatase would exhibit L-phenylalanine inhibition higher than 77%, by changing the substrate and inhibitor concentrations. The results in Table 5 illustrate that the employment of higher inhib-... 

Although a considerable literature records numerous studies in this field (A19, B17, C12, J3, L9, P16, S4, S5), many have been performed on preparations of tissues from other than human sources. In conformity with the subject of this chapter and to avoid species differences, most attention will be directed to human tissue alkaline phosphatases and in particular their variants. The stereospecific L-phenylalanine inhibition has provided the impetus to study its molecular mechanism, which necessarily requires an understanding of the mechanism of catalysis. It is expected that discovery of other stereospecific inhibitors will follow and that they may have even greater utility than L-phenylalanine. However, since it is the first such unique inhibitor, this section of the chapter will receive extensive treatment after a consideration of some basic kinetic information. 

Certain kinetic features of L-phenylalanine inhibition will now be described for the purified human intestinal and placental preparations of alkaline phosphatase. In experiments on the effect of pH, Ghosh and Fishman (G5) observed that the degree of stereospecific L-phenylalanine inhibition of alkaline phosphatase from rat or human intestine and from human placenta is highly pH-dependent. Rat or human intestinal alkaline phosphatase exhibited maximum inhibition at pH 9.2 with phenyl phosphate as substrate, whereas the human placental alkaline phosphatase had a peak at pH 9.6 (Fig. 10). 

The L-phenylalanine inhibition of rat (G5) or of (Fig. 12) human intestinal alkaline phosphatase and of human placental (G6) enzyme is of the uncompetitive type, because the double reciprocal plots of velocity and substrate concentration were all straight lines parallel to those obtained without the inhibitor. Consequently, the extent of the inhibition was greatly dependent on substrate (Fig. 11) and inhibitor concentrations (Fig. 10). Detailed studies have appeared elsewhere (G5). 

The use of the L-phenylalanine inhibition and heat sensitivity does serve (as illustrated in Cases A, B, C, and D) to separate into a separate category those sera whose biochemical patterns are abnormal in type compared to the pattern of a normal individual of the same blood tjqie, sex, and secretor status. If the heat sensitivity of the non-LPSAP is higher than expected, one has grounds to consider the presence of a bone phosphatase. Its amount can be quantitated readily. 

In the present state of knowledge and in the absence of information of the patient s diagnosis, one cannot yet expect to correctly identify a serum as mostly bone or mostly liver in origin with respect to its alkaline phosphatase by heat inactivation or L-phenylalanine inhibition. With the inclusion of additional studies, such as starch-gel electrophoresis, neuraminidase treatment, and continued study over a period of time, one can increase the certainty of the interpretation of the origin of the serum alkaline phosphatase in patients. 

F7. Fishman, W. H., and Ghosh, N. K., Influence of reagents reacting with metal, thiol and amino sites on catalytic activity and L-phenylalanine inhibition of rat intestinal alkaline phosphatase. Biochem. J. (1967) (in press). 

K25. Kreisher, J. H., Close, V. A., and Fishman, W. H., Identification by means of L-phenylalanine inhibition of intestinal alkaline phosphatase components separated by starch-gel electrophoresis of serum. Clin. Chim. Acta 11, 122-127 (1965). 

Studies of the effect of HT on affective states and behavior ) 2, 67, 129-132 cannot be discussed fairly in a short review, A role of HT metabolism in the mental retardation of phenylketonuriaremains unproved, although it has been noted that phenylalanine inhibits transport and hydroxylation of Tp3,... 

B. Excess phenylalanine inhibits tyrosinase the first step toward melanin production, thus resulting in hypopigmentation. Excess melanin leads to hyperpigmentation. Melatonin is a hormone involved in the sleep cycle. Excessive stimulation of tyrosinase would lead to more melanin and therefore hyperpigmentation. Para-hydroxyphenylpyruvate means less transamination and perhaps more tyrosine converted to melanin and hyperpigmentation. 

Minor metabolites that accumulate because of the alternate pathways utilized in phenylketonuria may normally serve either physiological or pharmacological roles in the nervous system. To aggravate the deficiency of tyrosine created by shunting into these minor pathways in PKU, phenylalanine inhibits tyrosine transport across biological membranes. In turn, this curtails the source of neuroactive tyrosine derivatives that can be synthesized, including tyramine, octopamine, and the catecholamines. One can speculate that such deficiencies could interfere with neurotransmitter action. 

The carboxylic acid analogues lacking the a-amino group are not activated by gramicidin S-synthetase. Hydrocinnamic acid, the analogue of phenylalanine, inhibits reaction 1 non-competitively. This compound most likely does not bind to the reaction center of GS 2 (2). Cyclopentane carboxylic acid and isocapronic acid show a very weak inhibition of the L-Pro resp. L-Leu activation. Carboxylic acid analogues of L-Orn (5-aminovaleric acid as well as 4-aminobutyric acid and 6-aminocapronic acid with a shortened or extended chain) do not appreciably affect reaction 4. 

D-phenylalanine, the sterioisomer of the naturally occurring L-phenylalanine, inhibits the PAL activity. Szkutnicka and Lewak (18) found that the application of this compound to germinating seeds caused an increase in the PAL level. They attributed this to the decrease in the production of cinnamic acid and compounds derived therefrom. Similar results have been obtained by Amrhein and Gerhardt (19) with the PAL-inhibitors, L-a-aminooxy-8-phenylpropionic acid and a-aminooxyacetic acid on gherkin seedlings. They found that the synthesis of hydroxycinnamic acids was inhibited and that the PAL level increased. This increase was suppressed when tr<2 s-cinnamic acid was applied. 

The accumulation of phenylalanine and its metabolites may interfere with the metabolism of other amino acids. Stein and Moore, and later Knox, showed that in phenylketonuric patients the amount of other amino acids in the plasma is decreased while phenylalanine accumulates. This interaction between the amino acids metabolism acquires particular significance in view of the mode of amino acid uptake in the brain. The investigators demonstrated that phenylalanine inhibits tyrosine uptake in the brain. Thus, in the presence of large amounts of phenylalanine, protein synthesis in the brain might be inhibited. Furthermore, because of the absence of tyrosine, the biosynthesis of well-known neuroregulators derived from tyrosine, such as norepinephrine and 3,4-dihydroxyphenylethyl-amine, could also be reduced in the brain. 

The other approach is the selective inactivation of specific isoenzymes. Placental alkaline phosphatase, for instance, is remarkably stable to heat inactivation. Incubation of the enzyme at 65 °C has no effect on its activity, unlike the other isoenzymes which are inactivated. Other isoenzymes can be differentiated by their stability in other conditions. For instance phenylalanine inhibits placental and intestinal isoenzymes but has little effect on the bone and liver isoenzymes. 

In contrast to the system of Shimura et al. (1964), intact protoplasts of strain 10716 require six amino acids for production of bacitracin and, for maximal yield, glucose is needed. Disrupted protoplasts are unable to synthesize the antibiotic (Snoke, 1961). With protoplasts, the six needed amino acids are L-cysteine, L-isoleucine, L-leucine, L-histidine, L-ornithine, and L-asparagine. Neither D-orni-thine nor D-asparagine can be utihzed directly and D-phenylalanine inhibits formation of bacitracin in the absence of L-phenylalanine. A peptide factor in soybean was found to be stimulatory for synthesis of bacitracin by whole cells provided that D-phenylalanine is present in the reaction mixture (Snoke, i960, 1961). In the absence of the D-amino acid, the peptide factor is inactive. 

Furthermore, by addition of appropriate amino acid analogs, it is possible to suppress formation of the peptide antibiotics with very httle alteration of protein synthesis. For example, D-phenylalanine inhibits production of bacitracin (Snoke and Cornell, 1964) threo-j8-phenylserine, j8-2-thienylserine, a-methyl-tryptophan, 5-methyltryptophane, and -fluorophenylalanine suppress tyrocidine synthesis (Mach et aL, I963) and a combination of norvaline, norleucine, and hydroxyproline depresses formation of gramicidin S (Winnick et al., I96I). 

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