Effect of sulfhydryl group modification on the activities of 5-oxo-L-prolinase.
5-Oxo-L-prolinase was isolated from rat kidney by a new procedure; highly active and apparently homogeneous enzyme was obtained in 50% yield after 1700-fold purification. The enzyme, which couples cleavage of ATP to ADP with that of 5-oxo-L-proline to L-glutamate, is uncoupled by Ca2+, Co2+, or excess Mn2+ as well as by replacement of adenosine 5'-triphosphate or of 5-oxo-L-proline by certain analogs as previously reported. The enzyme has Mr = 325,000 and is composed of 2 apparently identical subunits. It contains 27 sulfhydryl groups/monomer, 6 of which can be titrated in the native enzyme and 2 of which are required for catalysis. One of the sulfhydryl groups that can be titrated with 5,5'-dithiobis(2-nitrobenzoic acid) and N-ethylmaleimide can be protected against modification by ATP or inosine 5'-triphosphate. The findings suggest that at least 1 sulfhydryl group is at or close to the nucleoside triphosphate binding site and is involved in cleavage of NTP. 5'-p-Fluorosulfonylbenzoyl adenosine and 5'-p-fluorosulfonylbenzoyl inosine interact with the sulfhydryl group involved in NTPase activity and also with another amino acid residue of the enzyme. The data provide additional strong evidence that (a) the enzyme can bind 5-oxo-L-proline in the absence of NTP, and that it can bind NTP in the absence of 5-oxo-L-proline, and (b) the L-glutamate synthesis and NTP-cleaving activities are catalyzed by the same protein.
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000581:Amidohydrolases / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D003035:Cobalt / Q000494:pharmacology; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D007668:Kidney / Q000201:enzymology; D046911:Macromolecular Substances; D008345:Manganese / Q000494:pharmacology; D008970:Molecular Weight; D011752:Pyroglutamate Hydrolase / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D013438:Sulfhydryl Compounds / Q000378:metabolism
D046911:Macromolecular Substances; D013438:Sulfhydryl Compounds; D003035:Cobalt; D008345:Manganese; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D004228:Dithionitrobenzoic Acid; D000581:Amidohydrolases; D011752:Pyroglutamate Hydrolase; D005033:Ethylmaleimide; D002118:Calcium