PURIFICATION AND CHARACTERIZATION OF ADENINE PHOSPHORIBOSYLTRANSFERASE FROM SACCHAROMYCES-CEREVISIAE

Adenine phosphoribosyltransferase (APRT) from Saccharomyces cerevisiae was purified approximately 1500-fold. The enzyme catalyzes the Mg-dep endent condensation of adenine and 5-phosphoribosylpyrophosphate (PRPP ) to yield AMP. The purification procedure included anion exchange chr omatography, chromatofocusing and gel filtration, Elution of the enzym e from the chromatofocusing column indicated a pi value of 4.7. The mo lecular mass for the native enzyme was 50 kDa; however, upon electroph oresis under denaturing conditions two bands of apparent molecular mas s of 29 and 20 kDa were observed, We have previously reported the pres ence of two separate coding sequences for APRT, APT1 and APT2 in S. ce revisiae. The appearance of two bands under denaturing conditions sugg ests that, unlike other APRTs, this enzyme could form heterodimers. Th is may be the basis for substrate specificity differences between this enzyme and other APRTs. Substrate kinetics and product inhibition pat terns are consistent with a ping-pong mechanism. The k(m) for adenine and PRPP were 6 mu M and 15 mu M, respectively and the V-max was 15 mu mol/min. These kinetic constants are comparable to the constants of A PRT from other organisms. (C) 1997 Elsevier Science B.V.