DISTANCE RELATIONSHIPS BETWEEN THE CATALYTIC, GTP AND ADP REGULATORY SITES OF GLUTAMATE DEHYDROGENASE LABELED WITH FLUORESCENT NUCLEOTIDE ANALOGUES (5'-P-FLUOROSULFONYLBENZOYL-1, N(6)-ETHENOADENOSINE)

Bovine liver glutamate dehydrogenase was covalently modified by the fluorescent nucleotide analogues, 5'-p-fluorosulfonylbenzoyl-1,N('6)-ethenoadenosine (5'-FSB(epsilon)A) and 5'-p-fluorosulfonylbenzoyl-2-aza-1,N('6)-ethenoadenosine (5'-FSBa(epsilon)A). The incorporation was limited to 1.28 mol -SB(epsilon)A/mol subunit and 1.15 mol -SBa(epsilon)A/mol subunit. From the comparison of the kinetic and binding properties of the modified enzymes to native enzyme, it was concluded that 5'-FSB(epsilon)A and 5'-FSBa(epsilon)A modify a guanosine 5'-triphosphate (GTP) inhibitory site on glutamate dehydrogenase. The amino acid residues reacting with 5'-FSB(epsilon)A and 5'-FSBa(epsilon)A were identified. Enzyme modified by 5'-FSB(epsilon)A contained 0.95 mol modified tyrosine and 0.33 mol modified lysine, quantitatively accounting for the total incorporation prior to acid hydrolysis. As a function of time of incubation with 5'-FSB(epsilon)A, modification of tyrosine but not lysine correlates directly with the change in GTP inhibition. The peptides containing modified residues were isolated; tyrosine-262 was identified as the essential residue in the GTP site and in addition, at least two lysine modified peptides were observed, one of which contained lysine-245. In contrast, enzyme modified by 5'-FSBa(epsilon)A contained almost equal amounts of modified tyrosine and lysine as a function of time of incubation, suggesting that tyrosine and lysine are present in the GTP site and that 5'-FSBa(epsilon)A once bound, may react with either but not both. The distance between the catalytic, GTP inhibitory and adenosine 5'-diphosphate (ADP) activatory site was evaluated by energy transfer. A distance of 18 (ANGSTROM) was calculated between the GTP and ADP sites from the quenching of -SB(epsilon)A fluorescence upon titration with 2',3'-0-(2,4,6-trinitrocyclohexadienylidene)-ADP (TNP-ADP). A distance of 33 (ANGSTROM) was calculated between the catalytic site labeled with 4-iodo-acetamidosalicylic acid (ISA) and TNP-ADP at the ADP site. Enzyme labeled with 5'-FSBa(epsilon)A and ISA was used to calculate a distance of 23 (ANGSTROM) between the GTP and catalytic sites. The GTP and ADP sites are thus distinct and are closer to each other than to the catalytic site. The solution conformations of 5'-FSB(epsilon)A and fluorosulfonylbenzoyl derivatives of adenosine and guanosine were analyzed by fluorescence and proton nuclear magnetic resonance spectroscopy and may be a determinant of their reactions with enzymes.