Molecular physiology of phosphoryl group transfer from carbamoyl phosphate by a hyperthermophilic enzyme at low temperature.
Enzymes from thermophilic organisms often exhibit low activity at reduced temperature. To obtain a better understanding of this sluggishness, we have studied the reaction at 24 degrees C of the carbamate kinase (CK) from the hyperthermophile Pyrococcus furiosus. This enzyme is much slower at low temperature than is the CK from the mesophile Enterococcus faecalis. X-ray structures demonstrated bound ADP (even when no nucleotide was added) with the hyperthermophilic but not with the mesophilic CK. We use centrifugal gel filtration, rate of dialysis and pulse-chase experiments to demonstrate that the pyrococcal enzyme, at 24 degrees C, binds ADP avidly (K(D) = 34 nM), that ADP dissociates from this complex with a t1/2 value of 2.4 s, and that ADP binding is very fast (kappa = 8.4 x 10(6) M(-1) x s(-1)). The high affinity, rather than restrictions to dissociation, explains the isolation of the pyrococcal enzyme as an ADP complex. Carbamoyl phosphate adds quickly to this complex, and ADP cannot dissociate from the resulting ternary complex, being that it is converted very slowly (t1/2 = 10.3 s) to ATP, which dissociates quickly (t1/2 < 2.4 s). The slow conversion is a part of the normal enzyme reaction and limits the rate of the reaction at 24 degrees C. Thus, the sluggishness of the enzyme at low temperature is not due to slow substrate binding or product release but to the very slow rate of isomerization between enzyme-bound substrates and products. Probably the catalysis of the phosphoryl group transfer is less efficient at low temperature, as suggested by structural data showing that Lys131 is improperly positioned to assist the transfer.
Ramón-Maiques
Santiago
S
Instituto de Biomedicina de Valencia (CSIC), Jaume Roig 11, 46010-Valencia, Spain.
Britton
Hubert G
HG
Rubio
Vicente
V
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D002221:Carbamyl Phosphate / Q000378:metabolism*; D002384:Catalysis; D002850:Chromatography, Gel; D013293:Enterococcus faecalis / Q000201:enzymology*; D008958:Models, Molecular; D017851:Phosphotransferases (Carboxyl Group Acceptor) / Q000302:isolation & purification / Q000378:metabolism*; D020027:Pyrococcus furiosus / Q000201:enzymology*; D013696:Temperature
D002221:Carbamyl Phosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D017851:Phosphotransferases (Carboxyl Group Acceptor); C019288:carbamate kinase