Distance relationships between the catalytic site labeled with 4-(iodoacetamido)salicylic acid and regulatory sites of glutamate dehydrogenase

Abstract

The distance between the catalytic site on bovine liver glutamate dehydrogenase labeled with 4-(iodoacetamido)salicylic acid (ISA) and the adenosine 5'-diphosphate (ADP) activatory site occupied by the analogue 2',3'-O-(2,4,6-trinitrocyclohexadienylidene)adenosine 5'-diphosphate (TNP-ADP) was evaluated by energy transfer. Native enzyme and enzyme containing about 1 mol of acetamidosalicylate/mol of subunit bind about 0.5 mol of TNP-ADP/mol of subunit, and TNP-ADP competes for binding with ADP to native and modified enzyme, indicating that the analogue is a satisfactory probe of the ADP site. From the quenching of acetamidosalicylate donor fluorescence upon addition of TNP-ADP, an average distance of 33 A was determined between the catalytic and ADP sites. The fluorescent nucleotide analogue 5'-[p-(fluorosulfonyl)benzoyl]-2-aza-1,N6-ethenoadenosine (5'-FSBa epsilon A) reacts covalently with glutamate dehydrogenase to about 1 mol/peptide chain. As compared to native enzyme, the SBa epsilon A-enzyme exhibits decreased sensitivity to GTP inhibition but retains its catalytic activity as well as its ability to be activated by ADP and inhibited by high concentrations of NADH. Complete protection against decreased sensitivity to GTP inhibition is provided by GTP in the presence of NADH. It is concluded that 5'-FSBa epsilon A modifies a GTP site on glutamate dehydrogenase. The distance of 23 A between the catalytic site labeled with ISA and a GTP site labeled with 5'-FSBa epsilon A was measured from the quenching of salicylate donor fluorescence in the presence of the SBa epsilon A acceptor on a doubly labeled enzyme. The average distance between the ADP and GTP sites was previously measured as 18 A [Jacobson, M. A., & Colman, R. F. (1983) Biochemistry 22, 4247-4257], indicating that the regulatory sites of glutamate dehydrogenase are closer to each other than to the catalytic site.

Distance between the substrate and regulatory reduced coenzyme binding sites of bovine liver glutamate dehydrogenase by resonance energy transfer

“…Native and ISA-modified enzymes (each at 0.96 mg/ml) were compared at 303 nm and the difference in absorbance was used to measure the amount of ISA present. The incorporation was 0.69 -0.81 mol acetamidosalicylate/mol enzyme subunit, which is comparable to the value measured using radioactive reagent by Jacobson and Colman [3].…”

“…The value of I C~ can vary between 0-4 depending on the relative orientation of the donor and acceptor transition dipoles [l]. As reported by Jacobson and Colman [3], acetamidosalicylate bound to bovine glutamate dehydrogenase had a polarization value of 0.26. Haas et al [37] calculated that with a polarization less than 0.3 the uncertainty in K' would produce an error in the distance calculation of no more than 10%.…”

“…The concentration was determined from the absorbance at 302 and 268 nm using the absorptioncoefficientsof9.13 x lo3 M-'cm-' and 1.30 x lo4 M-' cm-respectively [23]. Bovine liver glutamate dehydrogenase, after dialysis against 0.1 M potassium phosphate pH 7.1, was modified under conditions similar to those described by Jacobson and Colman [3] with the following changes. A solution of 4 mM 4-(iodoacetamido)salicylic acid was prepared by slowing titrating ISA in water to pH 6.0 with 25 mM KOH.…”

“…5A, line 2). &Ado, like ISA-modified enzyme, has a maximum emission near 395 nm when excited at 303 nm [3].…”

“…The modified residue was isolated and identified as lysine '26 at the 2-oxoglutarate binding site [IS]. Once bound to the enzyme, acetamidosalicylate has a maximum emission at 395 nm when excited at 303 nm [3,17]. This chromophore has been used as a fluorescent probe to measure the distance between the 2-oxoglutarate site and the ADP activating site occupied reversibly by 2'(3')-0-(2,4,6-trinitrocyclohexadieny1idene)-adenosine 5'-diphosphate (TNP-ADP), and between the 2-oxoglutarate site and the GTP site labeled covalently by 5'-p-fluorosulfonylbenzoyl-2-aza-l, N6-ethenoadenosine [3].…”