Chemical modification of dCMP hydroxymethylase

Conference · Thu May 01 04:00:00 EDT 1986 · Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)

OSTI ID:5022934

Lee, M H; Gautam-Basak, M; Sander, E G

dCMP hydroxymethylase from E. coli B infected with a T-4 bacteriophage amber mutant is a dimer with a subunit molecular weight of 27,000 gm mol/sup -1/. Chemical modification of the homogeneous enzyme with the sulfhydryl reagents N-ethyl maleimide (NEM) and 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) leads to complete loss of activity. Aminopterin or methotrexate alone do not protect the enzyme against NEM inactivation but these compounds plus dCMP protect the enzyme far better than dCMP alone. Titration of the enzyme with /sup 14/C-NEM indicate that loss of enzyme activity correlates with sulfhydryl group modification. One (0.9 +/- 0.1) sulfhydryl per enzyme dimer causes complete inactivation. Titration of sulfhydryl groups with DTNB was monitored both by activity measurements and absorbance at 412 nm. About 90% of the enzyme activity was lost with the DTNB modification of one sulfhydryl group per dimer (t/sub 1/2/ = 1.0 min). The final 10% of enzyme activity loss requires the modification of a second sulfhydryl group which reacts at a much slower rate (t/sub 1/2/ = 20 min). The rates of sulfhydryl group modification by DTNB measured spectrophotometrically and the rates of enzyme activity loss are in excellent agreement. It would appear that two active site sulfhydryl groups per enzyme dimer are required for activity. The rapid modification of one sulfhydryl likely causes a conformational change which results in the slower reaction of the second sulfhydryl group.

• Research Organization: Texas A and M Univ., College Station
• OSTI ID: 5022934
• Report Number(s): CONF-8606151-
• Conference Information: Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States) Journal Volume: 45:6
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ANTIMITOTIC DRUGS
BACTERIA
BENZOIC ACID
BIOLOGICAL EFFECTS
CARBON 14 COMPOUNDS
CARBOXYLIC ACIDS
DRUGS
ENZYME ACTIVITY
ENZYMES
ESCHERICHIA COLI
ESTERASES
HYDROLASES
IMIDES
INACTIVATION
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
MICROORGANISMS
MONOCARBOXYLIC ACIDS
NEM
NUCLEASES
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PHOSPHODIESTERASES
RADIOSENSITIZERS
TRACER TECHNIQUES