Deoxycytidylate hydroxymethylase: purification, properties, and the role of a thiol group in catalysis.
Deoxycytidylate (dCMP) hydroxymethylase from Escherichia coli infected with a T-4 bacteriophage amber mutant has been purified to homogeneity. It is a dimer with a subunit molecular weight of 28,000. Chemical modification of the homogeneous enzyme with N-ethylmaleimide (NEM) and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) leads to complete loss of enzyme activity. dCMP can protect the enzyme against NEM inactivation, but the dihydrofolate analogues methotrexate and aminopterin alone do not afford similar protection. Compared to dCMP alone, dCMP plus either methotrexate or aminopterin greatly enhances protection against NEM inactivation. DTNB inactivation is reversed by dithiothreitol. For both reagents, inactivation kinetics obey second-order kinetics. NEM inactivation is pH dependent with a pKa for a required thiol group of 9.15 +/- 0.11. Complete enzyme inactivation by both reagents involves the modification of one thiol group per mole of dimeric enzyme. There are two thiol groups in the totally denatured enzyme modified by either NEM or DTNB. Kinetic analysis of NEM inactivation cannot distinguish between these two groups; however, with DTNB kinetic analysis of 2-nitro-5-thiobenzoate release shows that enzyme inactivation is due to the modification of one fast-reacting thiol followed by the modification of a second group that reacts about 5-6-fold more slowly. In the presence of methotrexate, the stoichiometry of dCMP binding to the dimeric enzyme is 1:1 and depends upon a reduced thiol group. It appears that the two equally sized subunits are arranged asymmetrically, resulting in one thiol-containing active site per mole of dimeric enzyme.
Lee
M H
MH
Department of Biochemistry and Biophysics, Texas A&M University, College Station 77843.
Gautam-Basak
M
M
Woolley
C
C
Sander
E G
EG
D004228:Dithionitrobenzoic Acid / Q000494:pharmacology*; D004926:Escherichia coli / Q000201:enzymology*; D005033:Ethylmaleimide / Q000494:pharmacology*; D019877:Hydroxymethyl and Formyl Transferases; D007700:Kinetics; D008970:Molecular Weight; D009579:Nitrobenzoates / Q000494:pharmacology*; D013438:Sulfhydryl Compounds; D013604:T-Phages / Q000201:enzymology*; D014166:Transferases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*
D009579:Nitrobenzoates; D013438:Sulfhydryl Compounds; D004228:Dithionitrobenzoic Acid; D014166:Transferases; D019877:Hydroxymethyl and Formyl Transferases; C100226:deoxycytidylate hydroxymethyltransferase; D005033:Ethylmaleimide