Km For Cofactor Research Articles

Replacement set mutagenesis of the four phosphate-binding arginine residues of thymidylate synthase.

Arginines R23, R178, R179 and R218 in thymidylate synthase (TS, EC 2. 1.1.45) are hydrogen bond donors to the phosphate moiety of the substrate, dUMP. In order to investigate how these arginines contribute to enzyme function, we prepared complete replacement sets of mutants at each of the four sites in Lactobacillus casei TS. Mutations of R23 increase K:(m) for dUMP 2-20-fold, increase K:(m) for cofactor 8-40-fold and decrease k(cat) 9-20-fold, reflecting the direct role of the R23 side chain in binding and orienting the cofactor in ternary complexes of the enzyme. Mutations of R178 increase K:(m) for dUMP 40-2000-fold, increase K:(m) for cofactor 3-20-fold and do not significantly affect k(cat). These results are consistent with the fact that this residue is an integral part of the dUMP-binding wall and contributes to the orientation and ordering of several other dUMP binding residues. Kinetic parameters for all R179 mutations except R179P were not significantly different from wild-type values, reflecting the fact that this external arginine does not directly contact the cofactor or other ligand-binding residues. R218 is essential for the structure of the catalytic site and all mutations of this arginine except R218K were inactive.

Regulation of recombinant rat tyrosine hydroxylase by dopamine.

Recombinant rat PC12 tyrosine hydroxylase, also called tyrosine 3-monooxygenase [L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2], purified from Escherichia coli is in an activated form with a low Km for the tetrahydrobiopterin cofactor and a pH optimum of 6.5. Pretreatment with low levels of the derived product, dopamine, inhibits catalytic activity, increases the Km for the cofactor, and shifts the pH curve towards a more acidic pH optimum. Labeled dopamine binds to tyrosine hydroxylase with high affinity (Kd = 1 microM) but low stoichiometry (r = 0.08 mol/mol of enzyme subunit). The binding of dopamine results in the appearance of a blue-green chromophore with lambda max at approximately 660 nm, which is consistent with the formation of a catecholamine-iron complex. In the absence of dopamine, the recombinant enzyme cannot be further activated by phosphorylation with cAMP-dependent protein kinase, although as much as 1 mol of phosphate is incorporated per mol of subunit. In contrast, the enzyme pretreated with dopamine is activated by phosphorylation in the same fashion and to the same extent as the native hydroxylase. The results suggest that the high-affinity binding of catecholamine products is a pivotal post-translational modification that determines the state of enzyme activation and the response to phosphorylation.

Changes in the cofactor binding domain of bovine striatal tyrosine hydroxylase at physiological pH upon cAMP-dependent phosphorylation mapped with tetrahydrobiopterin analogues

The structure of the cofactor binding domain of tyrosine hydroxylase (TH) was examined at physiological pH by determining kinetic parameters of (R)-tetrahydrobiopterin [(R)-BH4] and a series of tetrahydropterin (PH4) derivatives (6-R1-6-R2-PH4: R1 = H and R2 = methyl, hydroxymethyl, ethyl, methoxymethyl, phenyl, and cyclohexyl; R1 = methyl and R2 = methyl, ethyl, propyl, phenyl, and benzyl). A minimally purified TH preparation that was not specifically phosphorylated (designated as "unphosphorylated") was compared with enzyme phosphorylated with cAMP-dependent protein kinase. The Km for tyrosine with most tetrahydropterin analogues ranged between 20 and 60 microM with little decrease upon phosphorylation. Two exceptions were an unusually low Km of 7 microM with 6-ethyl-PH4 and a high Km of 120 microM with 6-phenyl-6-methyl-PH4, both with phosphorylated TH. Tyrosine substrate inhibition was elicited only with (R)-BH4 and 6-hydroxymethyl-PH4. With unphosphorylated TH (with the exception of 6-benzyl-6-methyl-PH4, Km = 4 mM) an inverse correlation between cofactor Km and side-chain hydrophobicity was observed ranging from a high with (R)-BH4 (5 mM) to a low with 6-cyclohexyl-PH4 (0.3 mM). An 8-fold span of Vmax was seen overall. Phosphorylation caused a 0.6-4-fold increase in Vmax and a 35-2000-fold decrease in Km for cofactor, ranging from a high of 60 microM with 6-methyl-PH4 to a low of 0.6 microM with 6-cyclohexyl-PH4. A correlation of the size of the hydrocarbon component of the side chain with affinity is strongly evident with phosphorylated TH, but in contrast to unphosphorylated enzyme, the hydroxyl groups in hydroxymethyl-PH4 (20 microM) and (R)-BH4 (3 microM) decrease Km in comparison to that of 6-methyl-PH4. Although 6,6-disubstituted analogues were found with affinities near that of (R)-BH4 (e.g., 6-propyl-6-methyl-PH4, 4 microM), they were frequently more loosely associated with phosphorylated TH than their monosubstituted counterparts (6-phenyl-PH4, 0.8 microM; cf. 6-phenyl-6-methyl-PH4, 8 microM). A model of the cofactor side-chain binding domain is proposed in which a limited region of nonpolar protein residue(s) capable of van der Waals contact with the hydrocarbon backbone of the (R)-BH4 dihydroxypropyl group is opposite to a recognition site for hydroxyl(s). Although interaction with either the hydrophilic or hydrophobic regions of unphosphorylated tyrosine hydroxylase is possible, phosphorylation by cAMP-dependent protein kinase appears to optimize the simultaneous operation of both forces.

Purification and characterization of rat striatal tyrosine hydroxylase. Comparison of the activation by cyclic AMP-dependent phosphorylation and by other effectors.

Purification of rat striatal tyrosine hydroxylase in the presence of protease inhibitors effected a high yield of apparently homogeneous enzyme which is stable to prolonged storage. The purified enzyme migrates as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular weight of 61,300. Removal of protease inhibitors results in the appearance of additional bands with molecular weights of 59,800 and 57,000. Cyclic AMP-dependent protein kinase incorporates 1 mol of phosphate/61,000-Da subunit of tyrosine hydroxylase, and concomitantly decreases the apparent Km of the enzyme for cofactor. Phosphorylated tyrosine hydroxylase is unstable at 37 degrees C, exhibiting a 50% decrease in apparent Vmax in 40 min with no change in Km for cofactor. Levels of incorporated phosphate remain constant over this time period. Tyrosine hydroxylase activated by and in the presence of phosphatidylinositol or high concentrations of NaCl exhibited a similar loss of activity at 37 degrees C, whereas enzyme activated by heparin was relatively stable. The rate of phosphorylation of tyrosine hydroxylase is markedly increased in the presence of any of these effectors, suggesting that they promote a common conformation. Further, heparin appears to bind to tyrosine hydroxylase at a site distant from the phosphorylation site. Physiological effectors of tyrosine hydroxylase may act in concert with cyclic AMP-dependent phosphorylation, perhaps by binding to an allosteric site, to regulate enzyme activity in vivo.

Effects of phospholipases on the kinetic properties of rat striatal membrane-bound tyrosine hydroxylase.

Rat striatal tyrosine hydroxylase can be isolated in both a soluble and a synaptic membrane-bound form. The membrane-bound enzyme, which exhibits lower Kms for both tyrosine (7 microM) and reduced pterin cofactor (110 microM) relative to the soluble enzyme (47 microM and 940 microM, respectively), can be released from the membrane fraction with mild detergent, and concomitantly its kinetic properties revert to those of the soluble enzyme. Treatment of membrane-bound tyrosine hydroxylase with C. perfringens phospholipase C increased the Km of the enzyme for tyrosine to 27 microM and the Vmax by 60% without changing the Km for cofactor. In contrast, treatment of membrane-bound tyrosine hydroxylase with V. russelli phospholipase A2 increased the Km for tyrosine to 48 microM, increased the Vmax, and increased the Km for cofactor to 560 microM. The enzyme remained bound to the membrane fraction following both phospholipase treatments. Addition of phospholipids to treated enzyme could partially reverse the effects of phospholipase A2 treatment, but not the effects of phospholipase C treatment. The kinetic properties of phospholipase-treated, detergent-solubilized tyrosine hydroxylase were identical to those of the control solubilized enzyme. Tyrosine hydroxylase appears to interact with synaptic membrane components to produce at least two separately determined consequences for the kinetic properties of the enzyme.

Tyrosine hydroxylase from bovine striatum: catalytic properties of the phosphorylated and nonphosphorylated forms of the purified enzyme.

The properties of purified tyrosine hydroxylase (TH) from bovine corpus striatum, both native and phosphorylated forms of the enzyme, were studied. TH had a tendency toward greater affinity for tetrahydrobiopterin (BH4) than for the synthetic cofactor 6-methyltetrahydropterin (6-MPH4), although the maximal velocity of the TH-catalyzed reaction was greater with 6-MPH4. Phosphorylation increased the affinity of TH for cofactor at pH 6.0, with little change in Vmax. At pH 7.0, phosphorylation caused increased activation of TH by increasing Vmax as well as reducing the Km for cofactor. The K1 for dopamine was increased twofold by phosphorylation at pH 6.0, but eightfold at pH 7.0. Phosphorylation was not associated with a change in Km for tyrosine at any pH or with any cofactor studied, although the Km for tyrosine of TH was cofactor-dependent and seven to eight times greater with 6-MPH4 than with BH4 as cofactor. Heparin and NaCl activated native TH at pH 6.0, but not at pH 7.0. Phosphorylated TH was unaffected by heparin or salt at pH 6.0, but was relatively inhibited at pH 7.0. The data are presented in the context of the physiological environment of TH.

Strain differences in kinetic and thermal stability of two mouse brain tryptophan hydroxylase activities

Levels of forebrain serotonin (5-HT), tryptophan, 5-hydroxyindoleacetic acid (5-HIAA) and hydroxylase cofactor (BH4) were comparable in two experimental mouse strains (A/J and C57Bl/6J) despite 2-3-fold differences in vitro in the relative activities of forebrain and midbrain tryptophan-5-monooxygenase (TPOH; EC 1.14.16.4). The enzyme activities did not differ with respect to Km for cofactor at saturating levels, but manifested different degrees of cooperativity with respect to cofactor when examined with BH4 concentrations within a physiological range. They differed also in the frequency and amplitude of kinetic variation around comparable mean velocity slopes across cofactor and time; in resistance to pre-incubation inactivation and responsiveness to its facilitation by calcium; in molecular weight heterogeneity as reflected in the distribution of molecular weight forms by gel diffusion chromatography; and in the number of peaks in power spectral analysis of kinetic variation patterns. Although the potential roles of small-molecule ligand and/or regulator proteins have not been ruled out, we hypothesize that differences in conformational stability underlie the differences in regulatory properties and make one enzyme activity more vulnerable to occlusive influences in vivo.

Effect of sodium dodecyl sulfate on the kinetic properties and molecular weight of rat striatal tyrosine hydroxylase

Soluble rat striatal tyrosine hydroxylase is activated by low concentrations (0.006–0.008%) of the anionic detergent sodium dodecyl sulfate (SDS). After treatment with the detergent, the enzyme exhibits a Km for cofactor decreased by an order of magnitude, and concamitantly loses the ability to be activated by the specific sulfated polysaccharide heparin (2). The identical effects of both effectors on the kinetic properties of the enzyme suggest that tyrosine hydroxylase is susceptible to specific conformational transitions which modulate its kinetic state.

Striatal tyrosine hydroxylases with high and low affinity for tyrosine: Implications for the multiple-pool concept of catecholamines

Synaptic membrane-bound tyrosine hydroxylase from rat striatum exhibits a decreased Km for cofactor and a decreased Km for substrate over values obtained for the soluble enzyme. The proximity of this fraction of the catecholamine biosynthetic apparatus to the probable site of neurotransmitter release and its higher affinity for substrate provide for a mechanism by which newly synthesized transmitter may be preferentially released.