Alphabeta Enzyme Research Articles

Mutational analysis of the subunit interface of Vibrio harveyi bacterial luciferase.

Bacterial luciferase is a heterodimeric (alphabeta) enzyme which catalyzes a light-producing reaction in Vibrio harveyi. In addition to the alphabeta enzyme, the beta subunit can self-associate to form a stable but inactive homodimer [Sinclair, J. F., Ziegler, M. M., and Baldwin, T. O. (1994) Nat. Struct. Biol. 1, 320-326]. The studies reported here were undertaken to explore the role of the subunit interface in the conformational stability of the enzyme. To this end, we constructed four mutant heterodimers in which residues at the subunit interface were changed in an effort to alter the volume of an apparent solvent accessible channel at the interface or to alter H-bonding groups. Equilibrium unfolding data for the heterodimer have been interpreted in terms of a three-state mechanism [Clark, C. A., Sinclair, J. F., and Baldwin, T. O. (1993) J. Biol. Chem. 268, 10773-10779]. However, we found that unfolding for the wild-type and mutant luciferases is better described by a four-state model. This change in the proposed mechanism of unfolding is based on observation of residual structure in the subunits following dissociation of the heterodimeric intermediate. All of the mutants display modest reductions in activity but, surprisingly, no change in the DeltaG2H2O value for subunit dissociation and no measurable change in the equilibrium dissociation constant relative to that of the wild-type heterodimer. However, the DeltaG1H2O value for the formation of the dimeric intermediate that precedes subunit dissociation is reduced for three of the mutants, indicating that mutations at the interface can alter the stability of a region of the alpha subunit that is distant from the interface. We conclude that the interface region communicates with the distal domains of this subunit, probably through the active center region of the enzyme.

Adenoviral gene therapy of the Tay-Sachs disease in hexosaminidase A-deficient knock-out mice.

The severe neurodegenerative disorder, Tays-Sachs disease, is caused by a beta-hexosaminidase alpha-subunit deficiency which prevents the formation of lysosomal heterodimeric alpha-beta enzyme, hexosaminidase A (HexA). No treatment is available for this fatal disease; however, gene therapy could represent a therapeutic approach. We previously have constructed and characterized, in vitro, adenoviral and retroviral vectors coding for alpha- and beta-subunits of the human beta-hexosaminidases. Here, we have determined the in vivo strategy which leads to the highest HexA activity in the maximum number of tissues in hexA -deficient knock-out mice. We demonstrated that intravenous co-administration of adenoviral vectors coding for both alpha- and beta-subunits, resulting in preferential liver transduction, was essential to obtain the most successful results. Only the supply of both subunits allowed for HexA overexpression leading to massive secretion of the enzyme in serum, and full or partial enzymatic activity restoration in all peripheral tissues tested. The enzymatic correction was likely to be due to direct cellular transduction by adenoviral vectors and/or uptake of secreted HexA by different organs. These results confirmed that the liver was the preferential target organ to deliver a large amount of secreted proteins. In addition, the need to overexpress both subunits of heterodimeric proteins in order to obtain a high level of secretion in animals defective in only one subunit is emphasized. The endogenous non-defective subunit is otherwise limiting.

Structure of bacterial luciferase beta 2 homodimer: implications for flavin binding.

The crystal structure of the beta 2 homodimer of Vibrio harveyi luciferase has been determined to 2.5 A resolution by molecular replacement. Crystals were grown serendipitously using the alpha beta form of the enzyme. The subunits of the homodimer share considerable structural homology to the beta subunit of the alpha beta luciferase heterodimer. The four C-terminal residues that are disordered in the alpha beta structure are fully resolved in our structure. Four peptide bonds have been flipped relative to their orientations in the beta subunit of the alpha beta structure. The dimer interface of the homodimer is smaller than the interface of the heterodimer in terms of buried surface area and number of hydrogen bonds and salt links. Inspection of the subunits of our structure suggests that FMNH2 cannot bind to the beta 2 enzyme at the site that has been proposed for the alpha beta enzyme. However, we do uncover a potential FMNH2 binding pocket in the dimer interface, and we model FMN into this site. This proposed flavin binding motif is consistent with several lines of biochemical and structural evidence and leads to several conclusions. First, only one FMNH2 binds per homodimer. Second, we predict that reduced FAD and riboflavin should be poor substrates for beta 2. Third, the reduced activity of beta 2 compared to alpha beta is due to solvent exposure of the isoalloxazine ring in the beta 2 active site. Finally, we raise the question of whether our proposed flavin binding site could also be the binding site for flavin in the alpha beta enzyme.

(Na+ + K+)-ATPase and plasma membrane polarity of intestinal epithelial cells: presence of a brush border antigen in the distal large intestine that is immunologically related to beta subunit.

The previously produced monoclonal antibody IEC 1/48 against cultured rat intestinal crypt cells (Quaroni, A., and K. J. Isselbacher. 1981. J. Natl. Cancer Inst. 67:1353-1362) was extensively characterized and found to be directed against the beta subunit of (Na+ + K+)-ATPase as assessed by immunological and enzymatic criteria. Under nondenaturing conditions the antibody precipitated the alpha-beta enzyme complex (98,000 and 48,000 Mr). This probe, together with the monoclonal antibody C 62.4 against the alpha subunit (Kashgarian, M., D. Biemesderfer, M. Caplan, and B. Forbush. 1985. Kidney Int. 28:899-913), was used to localize (Na+ + K+)-ATPase in epithelial cells along the rat intestinal tract by immunofluorescence and immunoelectron microscopy. Both antibodies exclusively labeled the basolateral membrane of small intestine and proximal colon epithelial cells. However, in the distal colon, IEC 1/48, but not C 62.4, also labeled the brush border membrane. The cross-reacting beta-subunit-like antigen on the apical cell pole was tightly associated with isolated brush borders but was apparently devoid of (Na+ + K+)-ATPase activity. Subcellular fractionation of colonocytes in conjunction with limited proteolysis and surface radioiodination of intestinal segments suggested that the cross-reacting antigen in the brush border may be very similar to the beta subunit. The results support the notion that in the small intestine and proximal colon the enzyme subunits are exclusively targeted to the basolateral membrane while in the distal colon nonassembled beta subunit or a beta-subunit-like protein is also transported to the apical cell pole.

Respiratory nitrate reductase from Paracoccus denitrificans

1. The b-type haem centres of the three (alpha, beta and gamma) subunit nitrate reductase from Paracoccus denitrificans have been analysed by redox potentiometry. Two components were identified with mid-point potentials +95 mV and +210 mV. 2. Washing, in the absence of Mg2+ ions, of cytoplasmic membrane vesicles from P. denitrificans promoted selective release of nitrate reductase activity. The released enzyme was purified by chromatography and shown to contain alpha and beta, but not gamma polypeptides. A haem spectrum was absent, consistent with the lack of the gamma subunit. The alpha and beta polypeptides of the water-soluble nitrate reductase had molecular masses that were identical to those of the detergent-purified enzyme and also of the nitrate reductase in cytoplasmic membranes. This observation, together with the failure of protease inhibitors to prevent release from the membrane, indicates that the release is not related to limited proteolysis of the alpha and/or beta polypeptides. The relative molecular mass of the water-soluble alpha beta enzyme was estimated to be approximately 200,000. 3. The water-soluble nitrate reductase was released from intact inverted cytoplasmic membrane vesicles as judged by loss of NADH-NO3- reductase activity and retention by the vesicles after washing of uncoupler-sensitive NADH-oxidase activity. These observations show that alpha and beta polypeptides, and therefore the active site for nitrate reduction, are located on the cytoplasmic side of the membrane. 4. Attempts to reverse the nitrate reductase activity of the enzyme, using nitrate as reductant plus ferricyanide or chlorate as tested oxidants, were unsuccessful. The implications for the mechanism of the enzyme are discussed.

The RNA-dependent DNA polymerase of avian sarcoma virus B77. Binding of viral and nonviral ribonucleic acids to the alpha, beta2, and alphabeta forms of the enzyme.

The extent of binding of various RNA species to the three forms of avian sarcoma virus B77 RNA-dependent DNA polymerase was determined using a sensitive nitrocellulose filter binding technique which was capable of detecting binding reactions with association constants as low as 3 X 10(6) liters X mole-1. All three enzyme forms, alphabeta, beta2, and alpha, bound to all single-stranded RNA species that were tested, including nonviral RNAs. 70 S viral RNA exhibited the highest association constant (about 10(11) liters X mole-1), and a population of virus-derived tRNA molecules from which tRNATrp had been removed, the lowest (about 3000 times lower). The affinity for other RNAs was roughly proportional to their size. The affinity of RNAs for the alphabeta enzyme form always exceeded that for the two others by a factor that depended on the particular RNA, never exceeded 6 and was sometimes as low as 1.2. The association constant of the alphabeta enzyme form with viral 70 S RNA was about 15-fold higher than that with viral 35 S RNA. 35 S RNA annealed to tRNATrp had an association constant that was only 2.5 times higher than that of 35 S RNA alone. This finding suggests that the tertiary structure of 70 S RNA plays a significant role in its affinity for B77 DNA polymerase.

RNA-dependent DNA polymerase of avian sarcoma virus B77. II. Comparison of the catalytic properties of the alpha, beta2, and alphabeta enzyme forms.

The alpha, beta2, and alphabeta forms of the RNA-dependent DNA polymerase of avian sarcoma virus B77 grown in duck embryo fibroblasts have been compared with respect to several kinetic properties. The following results were obtained. 1. The Km values for dTTP and dGTP for enzyme forms alpha, beta2, and alphabeta were 77, 39, and 74, and 6.8, 3.1, and 6.1 micronM, respectively. 2. The affinity of 70 S Rous sarcoma virus RNA for enzyme form alphabeta was about twice that for the other two forms. 3. The relative specific activities of the three enzyme forms on synthetic primer-templates such as poly(rA)-poly(dT) were almost the same. The viral 70 S RNA-dependent specific activities were 2 to 3 orders of magnitude lower and in the ratio of 1:3:5 for enzyme forms alpha:beta2:alphabeta. Addition of exogenous oligo(dT) stimulated the 70 S viral RNA-dependent activity of enzyme forms alphabeta and beta2 by a factor of 3, and that of enzyme form alpha by a factor of 30, so that it then became the most active transcriptase of viral 70 S RNA. 4. The largest transcripts formed by the three enzyme forms with 70 S viral RNA as primer-template were about 4,500 nucleotides long. About one-third of the total amount of polynucleotides polymerized by the alphabeta enzyme was in the form of such transcripts. This proportion was far higher than for the other two enzyme forms. 5. All three enzyme forms were capable of transcribing single-stranded into double-stranded DNA. 6. The 3-propylcyclohexyl piperidyl derivative of rifamycin SV, at a concentration of 100 microng/ml, inhibited enzyme forms beta2 and alphabeta by over 99.5 and 96%, respectively, but enzyme form alpha by only about 60%. 7. The beta2 and alphabeta forms of the enzyme were processive DNA polymerases, the alpha form a nonprocessive polymerase. 8. In general, these results indicate that in most respects the properties of the dimeric enzyme forms resemble each other much more closely than those of the alpha form. In some very important respects, such as affinity for viral RNA and the size of transcripts formed from it, the alphabeta enzyme form performs significantly better than either of the other two enzyme forms.

Further characterization of the Friend murine leukemia virus reverse transcriptase-RNase H complex

The purified reverse transcriptase-RNase H complex from Friend murine leukemia virus consists of a single polypeptide of 84,000 molecular weight, which after mild protease treatment in vitro or after intentional degradation during the purification procedure allows the generation of several additional polypeptides. Degradation destroys the RNA-dependent DNA polymerase activity with native RNA templates and reduces RNase H but does not affect response to synthetic template primers such as poly (rA)-Oligo (dT). The properties of the intact murine enzyme consisting of a single polypeptide of 84,000 molecular weight are compared to those of the avian alpha subunit and the avian alpha beta enzyme complex. The intact murine enzyme resembles the avian beta-containing enzyme complex and is different from alpha in the following respects: (i) it binds to native RNA templates; (ii) it transcribes native RNA templates into DNA, a reaction which can be inhibited by actinomycin D; (iii) RNase H activity behaves like a processive exonuclease; and (iv) analysis of the RNase H digestion products reveals oligonucleotides approximately four bases in length.

Sequence relatedness between the subunits of avian myeloblastosis virus reverse transcriptase.

One- and two-diminsional tryptic and chymotryptic peptide maps of 125-I-labeled alpha and alphabeta avian myeloblastosis virus DNA polymerase demonstrate that the alpha polypeptide of the one and two subunit enzymes are structurally similar, if not identical. Furthermore, the beta subunit contains the same major 125I-labeled peptides as alpha, plus several additional peptides. These relationships and the fact that aging of purified alphabeta avian myeloblastosis virus DNA polymerase increases the proportion of alpha DNA polymerase that can be isolated from the alphabeta enzyme by phosphocellulose chromatography, suggests that alpha is derived from beta by proteolytic cleavage.