Endonuclease and helicase activities of bacteriophage lambda terminase: changing nearby residue 515 restores activity to the gpA K497D mutant enzyme.

Terminase, the DNA packaging enzyme of bacteriophage lambda, is a heteromultimer of gpNu1 and gpA subunits. In an earlier investigation, a lethal mutation changing gpA residue 497 from lysine to aspartic acid (K497D) was found to cause a mild change in the high-affinity ATPase that resides in gpA and a severe defect in the endonuclease activity of terminase. The K497D terminase efficiently sponsored packaging of mature lambda DNA into proheads. In the present work, K497D terminase was found to have a severe defect in the cohesive end separation, or helicase, activity. Plaque-forming pseudorevertants of lambda A K497D were found to carry mutations in A that suppressed the lethality of the A K497D mutation. The two suppressor mutations identified, A E515G and A E515K, affected residue 515, which is located near the putative P-loop of gpA. A codon substitution study of codon 515 showed that hydrophobic and basic residues suppress the K497D defect, but hydrophilic and acidic residues do not. The E515G change was demonstrated to reverse the endonuclease and helicase defects caused by the K497D change. Moreover, the gpA K497D E515G enzyme was found to have kinetic constants for the high-affinity ATPase center similar to those of the wild type enzyme, and the endonuclease activity of the K497D E515G enzyme was stimulated by ATP to an extent similar to the ATP stimulation of the endonuclease activity of the wild type enzyme.

Selective proteolysis of the protein X subunit of the bovine heart pyruvate dehydrogenase complex. Effects on dihydrolipoamide dehydrogenase (E3) affinity and enzymic properties of the complex.

Selective proteolysis of the protein X subunit of native bovine heart pyruvate dehydrogenase complex may be accomplished without loss of overall complex activity. Partial loss of function occurs if Mg2+ and thiamin pyrophosphate are not present during proteinase arg C treatment as these cofactors are necessary to prevent cleavage of the E1 alpha subunit. Specific degradation of component X leads to marked alterations in the general enzymic properties of the complex. Lipoamide dehydrogenase (E3) exhibits a decreased affinity for the core assembly and the complex is much more susceptible to inactivation at high ionic strength. The inactive form of the complex is not readily re-activated by removal of salt. It appears that intact protein X and specifically the presence of its cleaved lipoyl domain is not essential for maintenance of an enzymically active pyruvate dehydrogenase complex. However, this protein has an important structural role in promoting the correct association of E3 with the E2 core assembly, an interaction that is required for optimal catalytic efficiency of the complex.

Topography of succinate dehydrogenase in the mitochondrial inner membrane. A study using limited proteolysis and immunoblotting.

The arrangement of the large (70,000-Mr) and small (30,000-Mr) subunits of succinate dehydrogenase in the mitochondrial inner membrane was investigated by immunoblot analysis of bovine heart mitochondria (right-side-out, outer membrane disrupted) or submitochondrial particles (inside-out) that had been subjected to surface-specific proteolysis. Both subunits were resistant to proteinase treatment provided that the integrity of the inner membrane was preserved, suggesting that neither subunit is exposed at the cytoplasmic surface of the membrane. The bulk of the small subunit appears to protrude into the matrix compartment, since the 30,000-Mr polypeptide is degraded extensively during limited proteolysis of submitochondrial particles without the appearance of an immunologically reactive membrane-associated fragment: moreover, a soluble 27,000-Mr peptide derived from this subunit is observed transiently on incubation with trypsin. Similar data obtained from the large subunit suggest that this polypeptide interacts with the matrix side of the inner membrane via two distinct domains; these are detected as stable membrane-associated fragments of 32,000 Mr and 27,000 Mr after treatment of submitochondrial particles with papain or proteinase K, although the 27,000-Mr fragment can be degraded further to low-Mr peptides with trypsin or alpha-chymotrypsin. A stable 32,000-34,000-Mr fragment is generated by a variety of specific and non-specific proteinases, indicating that it may be embedded largely within the lipid bilayer, or is inaccessible to proteolytic attack owing to its proximity to the surface of the intact membrane, possibly interacting with the hydrophobic membrane anchoring polypeptides of the succinate: ubiquinone reductase complex.

Component X of mammalian pyruvate dehydrogenase complex: structural and functional relationship to the lipoate acetyltransferase (E2) component.

The lipoate acetyltransferase (E2, Mr 70,000) and protein X (Mr 51,000) subunits of the bovine pyruvate dehydrogenase multienzyme complex (PDC) core assembly are antigenically distinct polypeptides. However comparison of the N-terminal amino acid sequence of the E2 and X polypeptides reveals significant homology between the two components. Selective tryptic release of the 14C-labelled acetylated lipoyl domains of E2 and protein X from native PDC generates stable, radiolabelled 34 and 15 kDa fragments, respectively. Thus, in contrast to E2 which contains two tandemly-arranged lipoyl domains, protein X appears to contain only a single lipoyl domain located at its N-terminus.