Prephenate aminotransferase

Enzyme Assays. Procedures for the HPLC assay of prephenate aminotransferase (32), the spectrophotometric assay of shikimate dehydrogenase... 

Prephenate aminotransferase The specific activity of this enzyme in Supernatant I was 19.2 nmol/min/mg, and the total activity was retained in Supernatant II following centrifugation at 60,000g. The homogenized pellet (about 300 mg of protein) exhibited enzyme activity (specific activity of... 

A similar activity level was obtained in the deoxycholate, Triton X-100, and NP-40 extract preparations. Octyl glucoside and CHAPS extract preparations showed no detectable prephenate aminotransferase activity. When the hemoglobin step was used, there was no increase in the soluble activity recovered in the initial supernatant fraction, but the specific activity of the deoxycholate (the only detergent tried in this experiment) extract increased about tenfold. We would anticipate equally good results with use of Triton X-100 or NP-40 in combination with the hemoglobin step. 

Figure 3-5. Biosynthesis of salicylic acid. The enzymes involved in this pathway are (a) chorismate mutase (E.C. 5.4.99.5), (b) prephenate aminotransferase (E.C. 2.6.1.78 and E.C. 2.6.1.79), (c) arogenate dehydratase (E.C. 4.2.1.91), (d) phenylalanine ammonia lyase (E.C. 4.3.1.5), (e) presumed P-oxidation by a yet to be identified enzyme, (f) benzoic acid 2-hydroxylase, (g) isochorismate synthase (E. C. 5.4.4.2), and (h) a putative plant pyruvate lyase.

Figure 6 Proposed biosynthetic pathways from chorismate (37), prephenate (38), and arogenate (41) to Phe (1), Tyr (2), and Trp (43) in plants and microorganisms. ADT, arogenate dehydratase AS, anthranilate synthase CM, chorismate mutase HPPAT, p-hydroxyphenylpyruvate aminotransferase PDH, prephenate dehydrogenase PPAAT, prephenate aminotransferase PPYAT, phenylpyruvate aminotransferase.

Fig. 5. Pretyrosine pathway. An alternate route for synthesis of phenylalanine and tyrosine described in bacteria and fungi. Enzymes labeled (a), (b), and (c) denote prephenate aminotransferase, pretyrosine dehydratase, and pretyrosine dehydrogenase, respectively.

Chorismate mutase 2 prephenate aminotransferase 3 prephenate dehydratase 4 prephenate dehydrogenase 5 arogenate dehydrogenase 6 phenylalanine aminotransferase 7 tyrosine aminotransferase 8 tyrosine 3-monooxygenase 9 phenylalanine 4-monooxygenase (C 2.6.5)... 

The CM-1 and CM-2 isozymes of chorismate mutase in N. silvestris have been shown to exist in plastidial and cytosolic compartments. Similar evidence also exists in support of a parallel compartmentation of DAHP synthase isozymes, DAHP synthase-Mn being plastid-localized and DAHP synthase-Co being cytosolic (d Amato and Ganson, unpublished data), Prephenate aminotransferase activity of N. silvestris is also largely or entirely localized within plastids (d Amato and Bonner, unpublished data). 

Results similar to those obtained with shikimate dehydrogenase were obtained with prephenate aminotransferase (Fig. 7). High levels of enzyme activity that were present in stationary phase declined upon dilution into fresh medium, reaching a low point during exponential phase. 

Fig. 7. Variation of the levels of prephenate aminotransferase as a function of growth phase. After 3.5 days (indicated by arrow) a portion of the cell culture monitored in the top panel was diluted 5-fold into fresh medium to initiate the culture shown in the 2nd panel. Subsequent transfers were carried out at 3 days and 4 days, respectively, to yield the cultures shown in panels 3 and 4 (bottom). L, E, and S denote lag, exponential and stationary phases of growth.

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