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K E Neet

Publications and source records attributed to K E Neet.

At least 73 records · Page 4Linked to original sources
PubMed1978

Threonine inhibition of the aspartokinase--homoserine dehydrogenase I of Escherichia coli. Threonine binding studies.

Both activities of the aspartokinase--homoserine I (AK-HSD) of Escherichia coli are inhibited by threonine. Careful threonine binding studies have now been done which have allowed us to distinguish the various effects of threonine on the enzyme. The ultrafiltration technique of H. Paulus ((1969) Anal. Biochem. 32, 101) for measuring ligand binding was shown to be comparable with equilibrium dialysis techniques. Reduction in error by utilization of this procedure enabled us to obtain evidence for two different sets of threonine sites by direct binding studies. The binding data were mathematically consistent with two independent classes of threonine sites, each of which contained four sites per tetramer and had a Hill coefficient of about 2.3--2.5. KD for the second set of sites was five- to tenfold greater than the high affinity sites, depending upon conditions. The data now suggest that the sequential model for site--site interactions adequately describes the cooperativity of threonine binding to the high affinity set of sites.

Aspartokinase Homoserine Dehydrogenase↗
PubMed1978

Role of arginyl residues in yeast hexokinase PII.

Yeast hexokinase PII is rapidly inactivated (assayed at pH 8.0) by either butanedione in borate buffer or phenylglyoxal, reagents which are highly selective for the modification of arginyl residues. MgATP alone offers no protection against inactivation, consistent with low affinity of hexokinase for this nucleotide in the absence of sugar. Glucose provides slight protection against inactivation, while the combined presence of glucose and MgATP gives significant protection, suggesting that modified arginyl residues may lie at the active site, possibly serving to bind the anionic polyphosphate of the nucleotide in the ternary enzyme:sugar:nucleotide complex. Extrapolation to complete inactivation suggests that inactivation by butanedione correlates with the modification of 4.2 arginyl residues per subunit, and complete protection against inactivation by the combined presence of glucose and MgATP correlates with the protection of 2 to 3 arginyl residues per subunit. When the modified enzyme is assayed at pH 6.5, significant activity remains. However, modification by butanedione in borate buffer abolishes the burst-type slow transient process, observed when the enzyme is assayed at pH 6.5, to such an extent that after extensive modification the kinetic assays are characterized by a lag-type slow transient process. But even after extensive modification, hexokinase PII still demonstrates negative cooperativity with MgATP and is still strongly activated by citrate when assayed at pH 6.5.

Arginine↗
PubMed1977

Relaxation spectra of yeast hexokinases. Isomerization of the enzyme.

Yeast hexokinase isozymes P1 and P11 exhibit a pH dependent, rapid relaxation process at 15 degrees C at enzyme concentrations of 100-474 muM and over a pH range of 6-8. The process was detected by equilibrium temperature jump spectroscopy using the indicator probe phenol red. The value of 1/tau varies from about 6 ms-1 at pH 8 for both isozymes to 50 ms-1 for P1 and 85 ms-1 for P11 at pH 6. The data are consistent with a mechanism involving an enzyme isomerization coupled to an ionization. The forward rate constant for the isomerization of the proposed mechanism varies between 3 and 7 ms-1; the ratio of the reverse rate constant to the ionization Ka is between 0.5 and 2 X 10(11) M-1 S-1; the estimated pKa varies between 5.5 and 6.1. The ranges of values in rate constants and pKa represent variations observed between preparations of the same isozyme and between isozymes. The isomerization rate is at least 50 times faster than catalysis under all conditions and the pKa is lower than that controlling activity. The rate of isomerization is unchanged by addition of sugar and nucleotide ligands, but the amplitude of the process is perturbed. These data imply that isomerizing and ionizing forms are sensitive to events at the active site. These equilibria between forms of hexokinase are fast enough, and have the right properties, to be important to the mechanism and regulation of the enzyme.

Adenosine Triphosphate↗
PubMed1976

pH-dependent effects of Cr(NH3)2ATP on kinetics of yeast hexokinase PII. Relationship to the slow transition mechanism.

The effect of Cr(NH3)2ATP, a virtually inert, inner sphere metal-ligand complex, on the kinetics of purified yeast hexokinase PII has been studied at pH 6.5 and pH 7.5. At pH 6.5, where the normal assays exhibit a slow burst-type transient, low concentrations of Cr(NH3)2ATP were found to activate both phii, the initial velocity, and phiII, the steady state velocity. At higher concentrations, Cr(NH3)2ATP was found to be a competitive inhibitor versus MgATP for both phii and phiII. The apparent Ki values for both velocities were the same. The inhibition by Cr(NH3)2ATP at pH 6.5 was found to be a slow process with half-times similar to those found for the normal burst-type transient at this pH value. At pH 7.5, where normal assays exhibit linear progress curves, Cr(NH3)2ATP behaved similarly to that observed before at pH 7 (Danenberg, D. D., and Cleland, W. W. (1975) Biochemistry 14, 28-39), i.e. it was a competitive inhibitor versus MgATP and it caused a slowing of the reaction rate over the first several minutes. The apparent Ki for the initial velocity was 8-fold higher than the apparent Ki for the steady state velocity, suggesting tighter binding of Cr(NH3)2ATP with time. Preincubation experiments indicated that the normal pH 6.5 burst-type transient could be eliminated by appropriate preincubation with Cr(NH3)2ATP and a sugar. In agreement with Danenberg and Cleland (1975), similar preincubations have been shown to produce linear assays at pH 7.5 in the presence of Cr(NH3)2ATP. Similar results were seen with MgITP as the nucleotide substrate, where a burst-type transient is not seen at either pH value under normal assay conditions. At pH 7.5, a slow decrease in the reaction rate is seen over the first several minutes in the presence of Cr(NH3)2ATP. The apparent Ki for phii was 7-fold higher than the apparent Ki value for phiII, again suggesting a tighter binding of Cr(NH3)2ATP with time. A similar observation was made at pH 6.5, but the Ki values for phii and phiII were the same, suggesting no tightening of the binding of Cr(NH3)2ATP with time at this pH value. These results suggested that both slow processes reflect the same basic molecular change, but the consequences are different at the two pH values, presumably because of the difference in the charge of the enzyme. The Cr(NH3)2ATP kinetics at pH 6.5 have been interpreted in terms of a modification of the slow transition mechanism for hexokinase (Shill, J. P., and Neet, K. E. (1975) J. Biol. Chem. 250, 2259-2268). It is postulated that glucose and Cr(NH3)2ATP induce the same slow conformational change at pH 6.5 as that induced by glucose and MgATP, which gives rise to the normal burst-type transient. This suggests that Cr(NH3)2ATP may be a useful tool for physical studies to determine the cause of the slow transition of yeast hexokinase. Activation by low concentrations of Cr(NH3)2ATP was interpreted as binding of the nucleotide to an activator site on the enzyme, causing a shift in the distribution of enzyme towards the more active form.

Adenosine Triphosphate↗