Dicyclohexylcarbodiimide modification of bovine heart mitochondrial transhydrogenase.

The hydrophobic carbodiimide dicyclohexylcarbodiimide (DCCD) has been shown to inhibit the catalytic (C) subunit of adenosine cyclic 3',5'-phosphate dependent protein kinase (EC 2.7.1.3) in a time-dependent, irreversible manner. The rate of inactivation was first order and showed saturation kinetics with an apparent Ki of 60 microM. Magnesium adenosine 5'-triphosphate (MgATP) was capable of protecting against this inhibition, whereas neither a synthetic peptide substrate nor histone afforded protection. Mg alone afforded some protection. When the catalytic subunit was aggregated with the regulatory subunit in the holoenzyme complex, no inhibition was observed. The inhibition was enhanced at low pH, suggesting that a carboxylic acid group was the target for interaction with DCCD. On the basis of the protection studies, it is most likely that this carboxylic acid group is associated with the MgATP binding site, perhaps serving as a ligand for the metal. Efforts to identify the site that was modified by DCCD included (1) modification with [14C]DCCD, (2) modification by DCCD in the presence of [3H]aniline, and (3) modification with DCCD and [14C]glycine ethyl ester. In no case was radioactivity incorporated into the protein, suggesting that the irreversible inhibition was due to an intramolecular cross-link between a reactive carboxylic acid group and a nearby amino group. Differential peptide mapping identified a single peptide that was consistently lost as a consequence of DCCD inhibition. This peptide (residues 166-189) contained four carboxylic acid residues as well as an internal Lys.(ABSTRACT TRUNCATED AT 250 WORDS)

Section: Discussionmentioning

confidence: 99%

Inhibition of the catalytic subunit of cAMP-dependent protein kinase by dicyclohexylcarbodiimide

Toner-Webb¹,

Taylor²

1987

“…residue modifiers has revealed the possible presence in TH of arginyl (Djavadi-Ohaniance & Hatefi, 1975), cysteinyl (Earle et al, 1978;, lysyl (M. Yamaguchi and Y. Hatefi, unpublished results), and carboxyl (Phelps & Hatefi, 1981, 1984bPennington & Fisher, 1981) groups whose modifications result in enzyme inactivation and are prevented by the presence of one or both substrates. Among these, a putative carboxyl group modified by DCCD1 appears to be at or near the NAD(H) binding site of TH, because (a) NAD(H) and analogues (5'-AMP and 5'-ADP), but not NADP(H) and analogues (2'-and 3'-AMP), protect TH against modification by DCCD and (b) unlike the unmodified TH, the DCCD-treated enzyme does not bind to NADagarose (Phelps & Hatefi, 1984a).…”

mentioning

confidence: 98%

Mitochondrial nicotinamide nucleotide transhydrogenase: active site modification by 5'-[p-(fluorosulfonyl)benzoyl]adenosine

Phelps¹,

Hatefi²

1985

Membrane-bound and purified mitochondrial energy-linked nicotinamide nucleotide transhydrogenase (TH) was inhibited by incubation with 5'-[p-(fluorosulfonyl)benzoyl]adenosine (FSBA), which is an analogue of TH substrates and their competitive inhibitors, namely, 5'-, 2'-, or 3'-AMP. NAD(H) and analogues, NADP, 5'-AMP, 5'-ADP, and 2'-AMP/3'-AMP mixed isomers protected TH against inhibition by FSBA, but NADPH accelerated the inhibition rate. In the absence of protective ligands or in the presence of NADP, FSBA appeared to modify the NAD(H) binding site of TH, because, unlike unmodified TH, the enzyme modified by FSBA under these conditions did not bind to an NAD-affinity column (NAD-agarose). However, when the NAD(H) binding site of TH was protected in the presence of 5'-AMP or NAD, then FSBA modification resulted in an inhibited enzyme that did bind to NAD-agarose, suggesting FSBA modification of the NADP(H) binding site or an essential residue outside the active site. [3H]FSBA was covalently bound to TH, and complete inhibition corresponded to the binding of about 0.5 mol of [3H]FSBA/mol of TH. Since purified TH is known to be dimeric in the isolated state, this binding stoichiometry suggests half-of-the-sites reactivity. A similar binding stoichiometry was found earlier for complete inhibition of TH by [14C]DCCD [Phelps, D.C., & Hatefi, Y. (1984) Biochemistry 23, 4475-4480]. The active site directed labeling of TH by radioactive FSBA should allow isolation of appropriate peptides for sequence analysis of the NAD(H) and possibly the NADP(H) binding domains.

“…The amino acid sequences of the two subunits of the Escherichia coli transhydrogenase have also been reported (Clarke et al, 1986). The bovine transhydrogenase is inhibited by a large number of protein modifying reagents, including thiol group modifiers (O'Neal & Fisher, 1977;Earle et al, 1978;Persson & Rydstrom, 1987), dicyclohexylcarbodiimide (DCCD) (Phelps & Hatefi, 1981, 1984aPennington & Fisher, 1981;Wakabayashi & Hatefi, 1987b), 7V-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ) (Phelps & Hatefi, 1984b), [(p-fluorosulfonyl)benzoyl]-5'-adenosine (FSBA) (Phelps & Hatefi, 1985; Wakabayashi & Hatefi, 1987a), ethoxyformic anhydride (Yamaguchi & Hatefi, 1985), dansyl chloride (Yamaguchi & Hatefi, 1985), tetranitromethane (Wu & Fisher, 1982), pyridoxal phosphate (Yamaguchi & Hatefi, 1985), butanedione and phenylglyoxal (Djavadi-Ohaniance & Hatefi, 1975), and 4-chloro-7-nitrobenzofurazan (Nbf-Cl) (Persson et al, 1988). In addition, the enzyme activity is highly sensitive to trypsin (Juntti et al, 1970; Djavadi-Ohaniance & Hatefi, 1975;Blazyk et al, 1976).…”

mentioning

confidence: 99%

Mitochondrial nicotinamide nucleotide transhydrogenase: NADPH binding increases and NADP binding decreases the acidity and susceptibility to modification of cysteine-893

Yamaguchi¹,

Hatefi²

1989

The mitochondrial nicotinamide nucleotide transhydrogenase is a dimeric enzyme of monomer Mr 110,000. It is located in the inner mitochondrial membrane and catalyzes hydride ion transfer between NAD(H) and NADP(H) in a reaction that is coupled to proton translocation across the inner membrane. The amino acid sequence and the nucleotide binding sites of the enzyme have been determined [Yamaguchi, M., Hatefi, Y., Trach, K., & Hoch, J.A. (1988) J. Biol. Chem. 263, 2761-2767; Wakabayashi, S., & Hatefi, Y. (1987) Biochem. Int. 15, 915-924]. N-Ethylmaleimide, as well as other sulfhydryl group modifiers, inhibits the transhydrogenase. The presence of NADP in the incubation mixture suppressed the inhibition rate by N-ethylmaleimide, and the presence of NADPH greatly increased it. NAD and NADH had little or no effect. The NADPH effect was concentration dependent and saturable, with a half-maximal NADPH concentration effect close to the Km of the enzyme for NADPH. Study of the effect of pH on the N-ethylmaleimide inhibition rate showed that NADPH binding by the enzyme lowers the apparent pKa of the N-ethylmaleimide-sensitive group by 0.4 of a pH unit and NADP binding raises this pKa by 0.4 of a pH unit, thus providing a rationale for the effects of NADP and NADPH on the N-ethylmaleimide inhibition rate. With the use of N-[3H]ethylmaleimide, the modified sulfhydryl group involved in the NADP(H)-modulated inhibition of the transhydrogenase was identified as that belonging to Cys-893, which is located 113 residues upstream of the tyrosyl residue modified by [p-(fluorosulfonyl)benzoyl]-5'-adenosine at the putative NADP(H) binding site of the enzyme (see above references).(ABSTRACT TRUNCATED AT 250 WORDS)