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The nature of 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase in extracts of wild-type Neurospora crassa: A reaction controlled by two activating substrates and three allosteric negative modifiers

1968; Elsevier BV; Volume: 159; Issue: 2 Linguagem: Inglês

10.1016/0005-2744(68)90084-3

1878-1454

Colin H. Doy,

Enzyme Structure and Function

Abstract 1. 1. The 3-deoxy- d - arabino -heptulosonate 7-phosphate synthase (7-phospho-2-keto-3-deoxy- d - arabino -heponate) d -erythrose-4-phosphate-lyase (pyruvate-phosphorylating), EC 4.1.2.15) (DAHP synthase) of Neurospora crassa wild type 74A is a unit of physiological function but regultory differentiation. Kinetic measurements are made with crude extracts in order to minimise denaturation of possible unifying native molecular organisation. A number of tentative conclusions (see below) are reached regarding ligand-enzyme interactions. 2. 2. DAHP synthase is a thiol enzyme. Co 2+ stimulates variably, EDTA inhibits strongly and is reversed completely by Co 2+ . Between pH 6.2–8.0 there is little change from an optimal activity at about pH 6.5. An Arrhenius plot is linear without transition between 17° and 37°. Heat denaturation occurs above about 4 i °. ΔH ★ is approx. 16 000 cal/mole. Crude dialysed extracts are stable for at least 1 h at 37°, but are rapidly deactivated by dilution. 3. 3. The substrates erythrose 4-phosphate and phosphoenol pyruvate are also activators (positive modifiers) with strong cooperative interaction between a minimum of 2 active sites per substrate species ( m = 1.7–1.8). Apparent K m does not apply in these examples, but [ S ] 0·5 ( V ) for either substrate is about 1.8·10 −4 M. The reaction is presumptive ping-poing but includes cooperative site interactions. It is deduced that PEP adds first and the importance of PEP as an initiating metabolite of aromatic biosynthesis is discussed. 4. 4. At least three distinct kinds of active sites occur; those inhibited by phenylanine, those inhibited by tyrosine and those inhibited by tryptophan. The ratio between these is normally about 44:44:10. Inhibition by tryptophan has not previously been demonstrated at this site in Nature. These end products are negative modifers each showing strong cooperative interaction between at least two sites. They act in part by lowering cooperation between substrate molecules. One molecule of tyrosine may sometimes bind ( m = 0.9, [ M ] 0.5 = 6·10−6 M). The phenylalanine and tyrosine sites must partly interact since their inhibition pattern is synergistic. Inhibition by tyrosine and phenylalanine is noncompetitive with regard to each substrate. When m = 1.4 to 2, values of [ M ] 0.5 range from 8·10 −6 to 1.7·10 −5 for different negative modifiers. The enzyme is rapidly denatured at 45°. The phenylalanine-sensitive component is most labile, but selectively protected by phenylalanine. The tyrosine-sensitive component is selectively protected by phosphoenol pyruvate (PEP). The tryptophan-sensitive portion and a small non-inhibited portion survive for at least i h without protection. Heat denaturation does not create a non-inhibited portion.