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M Byrnes

Publications and source records attributed to M Byrnes.

7 recordsLinked to original sources
PubMed1995

Role of glycine 212 in the allosteric behavior of phosphofructokinase from Bacillus stearothermophilus.

Crystallographic studies indicate that the loop between alpha-helix 8 and beta-strand H (the 8H loop) which borders the effector site of Bacillus stearothermophilus phosphofructokinase (BsPFK) is involved in the allosteric mechanism of the enzyme [Schirmer, T., and Evans, P.R. (1990) Nature 343, 140-145]. The residue at one end of this loop, glycine 212, has been proposed to be a pivot about which the loop hinges. Using site-directed mutagenesis, glycine 212 was replaced with valine (G212V). Steady-state kinetic analysis and ligand binding studies on the altered and native PFKs showed that the G212V substitution resulted in discernible changes at the effector site. The mutated PFK required a 3-fold higher concentration of the allosteric inhibitor phosphoenolpyruvate than did the native enzyme to cause the same level of inhibition. The altered PFK had a 2-fold higher dissociation constant for the allosteric activator GDP than the wild-type enzyme. More importantly, whereas the native PFK was fully activated by 1 mM GDP from its PEP-inhibited T-state, the altered enzyme was only marginally activated. On the other hand, the G212V mutation resulted in no changes at the catalytic site of BsPFK. The catalytic rate constant kcat remained unchanged. The altered PFK had the same Km values for ATP and fructose-6-phosphate (Fru-6-P) as did the wild-type enzyme. Furthermore, starting from the same PEP-inhibited T-state, both enzymes gave identical sigmoidal responses to increasing Fru-6-P concentration, indicating that Fru-6-P can activate both to the R-state.

Allosteric Regulation↗
PubMed1994

Kinetic characteristics of phosphofructokinase from Bacillus stearothermophilus: MgATP nonallosterically inhibits the enzyme.

The kinetic mechanism of phosphofructokinase from Bacillus sterothermophilus has been investigated using steady-state measurements. The double-reciprocal patterns observed for initial velocity, product inhibition, and mixed alternate substrate studies of the reverse reaction establish that the mechanism involves rapid-equilibrium random binding of substrates and the formation of an abortive complex composed of enzyme, MgADP, and fructose 6-phosphate (E-MgADP-Fru-6P). Initial velocity patterns for the forward reaction show significant nonlinearity and resemble those seen for competitive substrate (MgATP) inhibition of an enzyme that obeys a random mechanism. A mutant BsPFK enzyme (GV212) was used to show that the inhibition is not due to MgATP binding in the effector site. Product and dead-end inhibition studies of the forward reaction are consistent with a random mechanism, after taking into account the effects of substrate inhibition by MgATP. Initial velocity measurements at low MgATP concentration show that the binding of MgATP is not a rapid-equilibrium process; i.e., the rate of catalysis is faster than the rate of substrate binding. It is concluded that the kinetic mechanism of the forward reaction is sequential random, with the rate of MgATP binding slower than the catalytic rate. A model is presented that incorporates these results and proposes that substrate binding proceeds through two alternative pathways, one of which is kinetically disfavored. The observed MgATP substrate inhibition arises from both reaction flux through the disfavored pathway and, to some extent, abortive binding of MgATP in the Fru-6P site.

Adenosine Diphosphate↗
PubMed1993

Site-directed mutagenesis of rabbit muscle phosphofructokinase cDNA. Mutations at glutamine 200 affect the allosteric properties of the enzyme.

Full-length cDNA for rabbit muscle phosphofructokinase has been cloned and characterized (Li, J., Chen, Z., Lu, L., Byrnes, M., and Chang, S. H. (1990) Biochem. Biophys. Res. Commun. 170, 1056-1060). The 2.8-kilobase cDNA was inserted in the plasmid vector pPL2 and transformed into Escherichia coli cells deficient in endogenous phosphofructokinase activity (DF 1020). The recombinant phosphofructokinase so prepared is nearly identical in kinetic properties and size of subunits to the enzyme isolated from rabbit muscle. On the basis of the sequence homology between the muscle and the bacterial phosphofructokinases and the crystallographic structure of the latter, the glutamine at position 200 of the muscle enzyme is implicated in the allosteric transitions. This residue was replaced by alanine (Q200A), glutamate (Q200E), or arginine (Q200R). The purified enzymes were analyzed for quaternary structure, activity, and allosteric properties. The native and all the altered enzymes are tetramers. At pH 7.0, the wild-type enzyme is sensitive to inhibition by ATP at concentration above 0.6 mM, and its activity responds to fructose 6-phosphate concentration cooperatively at high ATP concentration. In contrast, the mutated enzyme Q200R is virtually insensitive to ATP inhibition up to 7 mM. Thus at high ATP concentration, its activity responds to fructose 6-phosphate concentration is a manner similar to the activated form of the native enzyme. Under the same conditions, mutant Q200E exhibits cooperative behavior only at much higher concentration of fructose 6-phosphate. Mutant Q200A is active at pH 8.0 but inactive at pH 7.0. The native enzyme and all three mutants are activated by inorganic phosphate and fructose 2,6-bisphosphate and inhibited by citrate.

Allosteric Regulation↗
PubMed1993

Family planning practices among Lebanese, Turkish and Vietnamese women in Sydney.

This paper is based on some of the data collected in a fertility and family planning survey conducted in 1988 among a probability sample of 980 married immigrant women in Sydney. The sample consisted of 507 Lebanese, 250 Turkish and 223 Vietnamese women. This study has revealed differences within and between the 3 migrant groups and the general population. Fertility among the 3 groups, but particularly among the Lebanese, was relatively high. While most women understood about birth control, there was a heavy reliance on traditional methods. Incidence of sterilization and hysterectomy was low compared to the general population. Use of abortions as a method of birth control was quite common among the Turkish women. Most women relied on their doctors for contraceptive advice and there was little knowledge and even less use of family planning and women's health centres. The study revealed an unmet need for culturally sensitive family planning information and services to these women.

Adult↗
PubMed1990

Sequence diversity in the 5' untranslated region of rabbit muscle phosphofructokinase mRNA.

DNA sequences of two full-length rabbit muscle phosphofructokinase (RMPFK) cDNAs (A and B) show identical coding sequence but heterogeneous 5' untranslated regions. cDNA-A is formed by removal of a 1.7 kb upstream intron while cDNA-B retains the 3' region of this intron. A 2.8 kb upstream sequence of RMPFK gene contains several features characteristic of housekeeping genes: high GC content (67%) at its 5' end, with 50 CpG sites; five Sp1 sites; and no functional TATA box. Comparison of the 5' sequences of the two RMPFK cDNAs and three human muscle PFK cDNAs (Nakajima, H., et al., (1990) Biochem. Biophys. Res. Com. 166, 637) suggests that a single splicing event occurs involving different splicing donor sites but the same splicing acceptor site, resulting in diversity in the upstream sequence. These observations suggest that transcription of muscle PFK gene may start at multiple sites, another feature of housekeeping genes.

Animals↗