Human Glutamine Phosphoribosylpyrophosphate Amidotransferase

Human glutamine phosphoribosylpyrophosphate amidotransferase (amidophosphoribosyltransferase) exhibits a number of allosteric properties. One of these is interconversion between a small form, 133,000 daltons, and a large form, 270,000 daltons, following incubation with phosphoribosylpyrophosphate (PP-ribose-F’) and purine ribonucleotides, respectively. The present report has examined the kinetics of this interconversion process and correlated these findings with changes in enzyme activity. At pH 7.4, virtually 100% of the small form of amidophosphoribosyltransferase was converted to the large form following incubation with purine ribonucleotides for less than 5 min at 4”. In contrast, essentially none of the large form was converted to the small form following incubation with PP-ribose-P for 30 min at 4”. However, incubation with PI’-ribose-P for 5 min at 37” resulted in the conversion of more than 50% of the large to the small form. Hydrogen ion concentration was also shown to influence this interconversion process. The same conditions which produced 100% conversion of the small to the large form at pH 7.4, resulted in less than 50% conversion at pH 8.2. At pH 7.1, essentially none of the large form was converted to the small by the incubation conditions that yielded greater than 50% conversion at pH 7.4. The hydrogen ion concentration did not affect the apparent K,,, for PP-ribose-P, but the percentage inhibition produced by a given concentration of AMP or GMP was approximately twice as great at pH 7.1 as compared to pH 8.2. In addition, it was demonstrated that human amidophosphoribosyltransferase exhibited a lag phase before it attained maximal activity when assayed at 25”, and the duration of the lag phase was temperature-dependent. In the absence of purine ribonucleotides no lag phase was demonstrable. The lag phase could be eliminated by preincubation of the enzyme preparation with PP-ribose-P for 5 min at 37”. However, preincubation with PP-ribose-P for 30 min at

Human glutamine phosphoribosylpyrophosphate amidotransferase (amidophosphoribosyltransferase) exhibits a number of allosteric properties. One of these is interconversion between a small form, 133,000 daltons, and a large form, 270,000 daltons, following incubation with phosphoribosylpyrophosphate (PP-ribose-F') and purine ribonucleotides, respectively.
The present report has examined the kinetics of this interconversion process and correlated these findings with changes in enzyme activity. At pH 7.4, virtually 100% of the small form of amidophosphoribosyltransferase was converted to the large form following incubation with purine ribonucleotides for less than 5 min at 4". In contrast, essentially none of the large form was converted to the small form following incubation with PP-ribose-P for 30 min at 4". However, incubation with PI'-ribose-P for 5 min at 37" resulted in the conversion of more than 50% of the large to the small form. Hydrogen ion concentration was also shown to influence this interconversion process. The same conditions which produced 100% conversion of the small to the large form at pH 7.4, resulted i n less than 50% conversion at pH 8.2. At pH 7.1, essentially none of the large form was converted to the small by the incubation conditions that yielded greater than 50% conversion at pH 7.4.
The hydrogen ion concentration did not affect the apparent K,,, for PP-ribose-P, but the percentage inhibition produced by a given concentration of AMP or GMP was approximately twice as great at pH 7.1 as compared to pH 8.2.
In addition, it was demonstrated that human amidophosphoribosyltransferase exhibited a lag phase before it attained maximal activity when assayed at 25", and the duration of the lag phase was temperature-dependent.
In the absence of purine ribonucleotides no lag phase was demonstrable. The lag phase could be eliminated by preincubation of the enzyme preparation with PP-ribose-P for 5 min at 37". However, preincubation with PP-ribose-P for 30 min at 4" was not effective in eliminating the lag phase. The lag phase was also shown to be dependent on the hydrogen ion concentration.
The duration of the lag phase was greater at pH 7.1 than pH 8.2.
It is concluded from these studies that human amidophosphoribosyltransferase is a hysteretic enzyme. It is suggested that the slow conversion of the large to the small form of the enzyme is responsible for the lag phase observed in the enzyme assay.

Characterization
of Lag Phase -The results depicted in Fig. 1 (Fig. 2). While incubation with PP-ribose-P for 30 min at 4" did not eliminate the lag phase, incubation with PP-ribose-P for 5 min at 37" was effective in eliminating the lag phase. Incubation at 37" in the absence of PP-ribose-P had no effect on the duration of the lag phase.
The results shown in Fig. 3 illustrate that the duration of the lag phase was temperature-dependent, being greater at 25" than 30". When the enzyme was assayed at 37" no lag phase could be detected. Effect ofHydrogen Ion Concentration on the Interconversion Process ~ The effect of pH on the interconversion between the large and small forms of human amidophosphoribosyltransferase is illustrated in Fig. 7. At pH 7.1, as at pH 7.4, the small form was readily converted to the large form following incubation with AMP for 5 min at 4" (Fig. 7A)