Changes In Kinetic Constants Research Articles

Induction of Maize Glutathione S-Transferase by Herbicide Safener and their Effect on Enzyme Activity against Chlorsulfuron

The acetolactate synthase(ALS) activity, glutathione S-transferase(GST) activity and glutathione(GSH) content in maize was investigated in response to treatments with safener (S)-3-dichloroacetyl-2,2-dimethyl-4-ethyl-1,3-oxazolidine. When induced by the chiral compound, the GSH content in root and shoot of maize increased 91.10% and 71.66%, respectively, the recovery rate of ALS activity reached 77%, and the GST activity increased 103.77%. The activity of maize GST was also assayed towards chlorsulfuron in vitro; the results showed that the induction of GST activity against chlorsulfuron of 14.54% was obtained. The enhancement of GST activity was found to be concomitant with changes in kinetic constants, which was related directly to the enzyme affinity for the CDNB substrate.

Mandelamide hydrolase from Pseudomonas putida: characterization of a new member of the amidase signature family.

A recently discovered enzyme in the mandelate pathway of Pseudomonas putida, mandelamide hydrolase (MAH), catalyzes the hydrolysis of mandelamide to mandelic acid and ammonia. Sequence analysis suggests that MAH is a member of the amidase signature family, which is widespread in nature and contains a novel Ser-cis-Ser-Lys catalytic triad. Here we report the expression in Escherichia coli, purification, and characterization of both wild-type and His(6)-tagged MAH. The recombinant enzyme was stable, exhibited a pH optimum of 7.8, and was able to hydrolyze both enantiomers of mandelamide with little enantiospecificity. The His-tagged variant showed no significant change in kinetic constants. Phenylacetamide was found to be the best substrate, with changes in chain length or replacement of the phenyl group producing greatly decreased values of k(cat)/K(m). As with another member of this family, fatty acid amide hydrolase, MAH has the uncommon ability to hydrolyze esters and amides at similar rates. MAH is even more unusual in that it will only hydrolyze esters and amides with little steric bulk. Ethyl and larger esters and N-ethyl and larger amides are not substrates, suggesting that the MAH active site is very sterically hindered. Mutation of each residue in the putative catalytic triad to alanine resulted in total loss of activity for S204A and K100A, while S180A exhibited a 1500-fold decrease in k(cat) and significant increases in K(m) values. Overall, the MAH data are similar to those of fatty acid amide hydrolase and support the suggestion that there are two distinct subgroups within the amidase signature family.

Modification of presynaptic CaM kinase II affinity for ATP in hippocampus after long term blockade of serotonin reuptake

Ca 2+/calmodulin-dependent protein kinase II (CaMKII) is markedly enriched at synapses, where it is involved in the control of synaptic transmission, transmitter release and synaptic plasticity. CaMKII has also been found to be involved in the long-term action of antidepressants on post-receptor signaling mechanisms, because monoamine reuptake inhibitors induced an increase in autophosphorylation and activity of the kinase in nerve terminals of hippocampus. To study whether changes in the amount of enzyme or kinetic changes, due to posttranslational modifications, are responsible for kinase activation in nerve terminals, α–CaMKII level and kinetic constants of the autophosphorylation reaction as a function of ATP concentration were measured in presynaptic cytosol from hippocampus. Treatment with two serotonin reuptake inhibitors did not change the level of presynaptic kinase or the Vmax of autophosphorylation reaction. Instead the Km of the kinase for ATP was decreased 2.8-fold with fluvoxamine and 3.5-fold with paroxetine, implying an increase in the affinity for ATP. This result represents the first finding of changes in kinetic constants of a major brain enzyme after treatment with antidepressant drugs.

Analysis of wild-type and mutant aspartate aminotransferases using integrated rate equations

A general integrated rate equation was fit to reaction progress curves catalyzed by wild-type E. coli aspartate aminotransferase and the site-specific mutant enzymes, H193Q and Y70F. A nonlinear step-regression code, revised for this study selected from all kinetic constants in a general integrated rate equation for all unbranched enzyme mechanisms with stoichiometries upto two substrates and two products including terms for substrate inhibitions and that of an exogenous inhibitor. For each aspartate aminotransferase enzyme studied only kinetic constants consistent with a substituted enzyme mechanism were found statistically significant, thus the enzyme mechanism and sources of inhibition were determined objectively by statistics. The kinetic constants for wild-type and Y70F aspartate aminotransferase were similar to those previously reported indicating the validity of the integrated rate equation analysis. Minor changes in kinetic constants were observed for the H193Q mutant enzyme suggesting that the catalytic effects of the electrostatic hydrogen bonding network extending from the pyridine nitrogen of the cofactor through Asp-222, His-143, and His-189 ends prior to His-193.

Kinetic analysis of Cd uptake in Cd‐tolerant and intolerant populations of the moss Rhytidiadelphus squarrosus (Hedw.) Warnst and the lichen Peltigera membranacea (Ach.) Nyl.

SUMMARYCadmium uptake by photosynthetically Cd‐tolerant and intolerant populations of the moss Rhytidiadelphus squarrosus (Hedw.) Warnst and the lichen Peltigera membranacea (Ach.) Nyl. was investigated. Intolerant plants showed increased intra cellular Cd uptake compared to Cd‐tolerant material. Kinetic analysis indicated that reduced intracellular Cd uptake by Cd‐tolerant plant material was reflected in an increased Km or decreased Vmax for Cd. Pretreatment with KNO3 modified the extra cellular ion content of plant material, significantly reduced the apparent Km and could increase or decrease the Vmax for intracellular Cd uptake. Changes in kinetic constants caused by K pretreatment could be related to altered chemical equilibria when plant material was incubated in Cd solutions. Selective exclusion of Cd from intracellular locations was excluded as a tolerance mechanism in R. squarrosus and P. membranacea. Contrasting kinetic constants for intracellular Cd uptake in intolerant and Cd‐tolerant plant material were due to the prevailing extra cellular ion content when collected from the field. In R. squarrosus, K pretreatment resulted in increased uptake capacities and cell‐wall binding affinities for Cd in field‐grown moss apices. Laboratory‐grown apices, following K pretreatment, displayed increased uptake capacities but reduced affinities for Cd. Increased extra cellular Cd uptake in K‐pretreated moss apices was interpreted as additional weak electrostatic binding. Kinetic analysis indicated that electrostatic binding by extra cellular sites was less influential than the available supply of wall‐bounds ions on intracellular Cd uptake by moss apices.

Ontogenic and regional changes in kinetic constants of alpha-methyl-D-glucoside transport in chicken small intestine.

Ontogenic and regional changes in kinetic constants of alpha-methyl-D-glucoside transport in chicken small intestine.

Threonine 204 of the chaperone protein Hsc70 influences the structure of the active site, but is not essential for ATP hydrolysis.

The chaperone protein Hsc70 is an ATPase of unknown mechanism, although the crystal structure of the 44-kDa ATPase domain has been solved. This structure shows that the hydroxyl of threonine 204 is located close to the gamma-phosphate of ATP, in a position where it might be an intermediate phosphate acceptor in the hydrolysis reaction. We made two point mutations at residue 204 of Hsc70, threonine to valine (T204V) and threonine to glutamic acid (T204E). The wild-type ATPase domain had a Km for ATP of approximately 1 microM; the mutants had Km values of approximately 90 microM. The kcat values for the mutant proteins were also increased. After crystallization, the structures of the T204V and T204E proteins were solved and refined with data to 2.3- and 2.4-A resolution, respectively. The overall tertiary structure of the mutants showed little change from the wild type; however, significant changes were observed in the active site. Analysis of the structures suggested possible reasons for the changes in kinetic constants. Threonine 204 does not seem to be an obligatory intermediate phosphate acceptor in the hydrolysis reaction since the mutants retained appreciable ATPase activity.

Relations of enzymes inAspergillus repens grown under sodium chloride stress

Aspergillus repens, a salt-pan isolate, was halotolerant. When grown for 72 h (log phase) and 144 h (beginning of stationary phase) in a medium containing 2M sodium chloride, the activities of invertase, malate dehydrogenase (MDH), glucose-6-phosphate dehydrogenase (G6PDH), and glutamate dehydrogenase (GDH) were found to have increased. Control cultures grown in a medium devoid of 2M NaCl failed to show such changes. The activities of MDH, G6PDH, and GDH increased with rising concentrations of Na(+) (as NaCl) when added up to 100MM in vitro. At higher concentrations they decreased. Changes in kinetic constants, Km and Vmax of these enzymes, as well as their de novo synthesis, were found to be some of the responses to NaCl stress-mediated changes.

Microbial degradation kinetics of toxic organic chemicals over a wide range of concentrations in natural aquatic systems

AbstractThe degradation kinetics for several toxic organic compounds (p‐cresol, phenol, p‐ chlorophenol, acetone and methanol) exposed to natural aquatic microbial assemblages were examined over a wide range of concentrations. Compounds were added in radiolabeled form and incubated at 25°C for periods ranging from several hours to one day. The results revealed that degradation kinetics were multiphasic for each of the compounds during at least one sampling period, i.e., the kinetic parameters Kt and Vmax increased with increasing substrate concentration. The observed multiphasicity suggests the presence within the natural microbial assemblages of multiple uptake/degradative systems, each with distinguishable kinetic parameters and each dominating degradation at a different substrate concentration range. If the kinetic diversity is ignored and rates of degradation are calculated based on the assumption that kinetics follow simple hyperbolic (Michaelis‐Menten) relationships, then errors as large as 40,000‐fold can result. Thus, our results suggest that models for predicting the persistence of such compounds in natural waters should consider changes in kinetic constants with substrate concentration.

Renal Function and (Na+ + K+)-ATPase in Chronic Unilateral Hydronephrosis in Dogs

Recovery of various parameters of kidney function after varying periods of complete unilateral ureteral obstruction was studied in dogs under hydropenic conditions. Changes of PAH and inulin clearance appeared to be parallel. After one week of obstruction, renal clearances of PAH and inulin were decreased to seven per cent of values measured before the obstruction period, after two weeks to four per cent and after three or four weeks to two per cent. Within 10 to 28 days after release of obstruction by cutaneous ureterostomy, PAH and inulin clearance increased to 66 per cent after one week, to 50 per cent after two weeks, to 10 per cent after three weeks with no change after four weeks of obstruction. Na+ content in the hydronephrotic kidney differed from contralateral kidneys only in the inner medulla. The affinity for ouabain (dissociation constant, KD, normal = 3.85 × 10–9M; hydronephrotic = 3.05 ×10–9M; contralateral = 7.05 ×10–9M) was significantly higher only in the outer medulla of contralateral kidneys. Turnover number (normal = 3.6; hydronephrotic = 5.1; contralateral = 3.4 × 103min.−1) in hydronephrotic or contralateral outer medulla was not significantly different from normal. Changes in kinetic constants (association rate constant, k+1, normal = 4.49 × 104M−1sec.−1; hydronephrotic = 4.13 × 104M−1sec.−1; contralateral = 5.97 × 104M−1sec.−1; dissociation rate constant, k_1, normal = 1.03 × 10−4sec.−1; hydronephrotic = 1.29 × 10−4sec.−1; contralateral = 1.39 × 10−4sec.−1) were considered to be too small to be relevant. Similar changes of KD, k+1 and k−1 were observed in the renal cortex. The osmotic concentrating capacity correlated well with (Na+ + K+)-ATPase activity (r = 0.85) and number of 3H-ouabain binding sites (r = 0.89) in renal outer medulla. The results indicate that recovery of osmotic concentrating capacity depends on the length of obstruction, and that a reduction of (Na+ + K+)-ATPase molecules in the thick ascending limb of the loop of Henle is a primary factor in the decrease in the concentrating capacity of chronic unilateral hydronephrotic kidneys.

Effect of Succinylation on the Proteolysis of Food Proteins

ABSTRACTCasein, bovine serum albumin and soy globulin were succinylated and used as substrates, in both forms, for initial velocity studies of proteolysis by α‐chymotrypsin and pepsin. Succinylation resulted in modification of at least 91% of the free amine groups of the proteins. Proteolysis was determined by fluorometrically monitoring the appearance of amine groups. Kinetic constants were estimated by the direct equation plot using FORTRAN programs. Succinylation enhanced the initial proteolysis of all substrates by either enzyme as evidenced by an increased maximum velocity and decreased Michaelis constant for the modified substrates. Modification of casein caused the most pronounced differences in rates of proteolysis, resulting in the largest change in kinetic constants for pepsin and the smallest change for α‐chymotrypsin.

Insulin-like activity of proteases. V. Stimulation of cyclic adenosine 3',5'-monophosphate phosphodiesterase by an N-succinyl-L-alanyl-L-alanyl-L-alanine p-nitroanilide-hydrolyzing protease.

The stimulatory effect of an N-succinyl-L-alanyl-L-alanyl-L-alanine p-nitroanilidehydrolyzing protease (STA-protease), which was partially purified from Pronase, on cyclic adenosine 3', 5'-monophosphate (cyclic AMP) phosphodiesterase of bovine heart was investigated by measuring the changes in kinetic constants. The phosphodiesterase used showed hydrolytic activity towards cyclic AMP at a concentration of 1 or 100 μM. STA-protease stimulated low Km phosphodiesterase but not high Km phosphodiesterase activity, in the presence of ethylene glycol bis (β-aminoethylether)-N, N'-tetraacetic acid (EGTA). No stimulation of the low Km enzyme was observed when theophylline was used instead of EGTA. Proteases such as Pronase, trypsin, chymotrypsin, elastase, and substilisin BPN' stimulated the low Km enzyme to various extents. Although STA-protease showed the lowest caseinolytic activity among proteases tested, its stimulatory activity towards the low Km enzyme was rather high. STA-protease increased the Km and Vmax values of the low Km enzyme, whereas trypsin decreased the Km value without causing significant change in Vmax. The stimulatory mechanism of STA-protease probably differs from that of trypsin.

Cooperativity and the slow isomerization of deta-chymotrypsin.

δ-Chymotrypsin has previously been reported to undergo a slow isomerization between a high pH form and a neutral pH form (pK = 9) as observed by its reactivity towards a variety of substrates. [Garel, J-R and Labouesse, B. (1973) Eur. J. Biochem., 39, 293–300, Fersht, A. R. (1972) J. Mol. Biol. 64, 497–509] Since slow transitions, e.g., isomerizations, between two or more enzyme forms which have different kinetic properties can produce kinetic cooperativity for either monomeric or oligomeric enzymes [Ainslie, G. R., Jr., Shill, J. P. and Neet, K. E. (1972) J. Biol. Chem. 247, 7088–7096], we have investigated whether δ-chymotrypsin might show cooperativity with some substrates. Careful statistical analysis of the steady state kinetics has demonstrated that negative cooperativity with the substrate N-CBZ-l-tryptophan p-nitrophenyl ester occurs at pH 9 and 25 °C, with a Hill coefficient of about 0.6. Mechanisms which involve interactions between catalytic sites cannot be the source of the cooperativity of δ-chymotrypsin since it is monomeric and has only a single catalytic site. For these reasons the δ-chymotrypsin kinetics presented here have been interpreted in terms of the slow transition mechanism for kinetic cooperativity. 1. The negative cooperativity with the tryptophan substrate disappears if the temperature is decreased or the pH is lowered. This observation is in accord with the slow transition mechanism since the fraction of δ-chymotrypsin present in the high pH form is kinetically insignificant under these conditions. 2. Alterations in the nature of the substrate and ionic strength also eliminate the observation of negative cooperativity. Such a loss in cooperativity is consistent with the slow transition mechanism in which the cooperativity may be sensitive to small changes in kinetic constants.

Kinetic analysis of calcium activation of brain acetylcholinesterase forms

Calcium activation of acetylcholine hydrolysis by bovine brain acetylcholinesterase (Acetylcholine hydrolase, EC 3.1.1.7) forms has been analyzed in terms of changes in kinetic constants and thermodynamic activation parameters. De-acetylation was determined to be the major rate-influencing step in acetylcholine hydrolysis by both 60 000- and 240 000-dalton forms of the brain enzyme and 10 mM Ca 2+ increased the rate constant for this step ( +3) by approximately 30% for bot forms. For the smaller acetylcholinesterase form the effects of Ca 2+ on de-acetylation was equivalent to its effect on the overall rate constant ( K) and occurred without an effect on p K. In the case of the 240 000-dalton species, the overall rate constant was increased by Ca 2+ by 33% at pH 8.0 and 81% at pH 7.25 and involved a p K shift of −0.2 pH units. For both enzyme forms the rate constants for acetylation ( K +2) were increased by Ca 2+. Thermodynamic analysis suggested that Ca 2+ activation of the acetylation step was entropically driven. Differences between the two enzymes forms in terms of Ca 2+ appear to result from association of low molecular weight species.

Anaerobic stimulation of sugar transport in avian erythrocytes

The kinetic parameters of the sugar transport in avian erythrocytes were evaluated under aerobic and anaerobic conditions. In anaerobic cells, transport measurements with 3-O-[ 14 C] methylglucose resulted in a set of similar dissociation-like constants. Thus the Michaelis constants of 3-O-[ 14 C] methylglucose entry and exit, K so and K si , were 8 and 7 mM, respectively. The equilibrium exchange constant, B s , and the counterflow constant, R s , were 9 and 11 mM, respectively. The activity constant for 3-O- methylglucose transport, F s , defined as V/K m , was 4 ml/h per g. This set of kinetic constants was compatible with a symmetrical mobile-carrier model. In contrast, the Michaelis constant for glucose entry, K go , was 2 mM and less than the counterflow constant, R g (8 mM). This result could be accounted for by slower movement of the glucose-carrier complex than the free carrier. The activity constant for glucose transport, F g , was 5 ml/h perg. Under aerobic conditions, two of the dissociation-like constants ( K si and B s ) for 3-O- methylglucose transport were significantly larger than those obtained in anaerobic cells, but the remaining two ( K so and R s ) remained unchanged. The values, for K so , K si , B s and R s were 8, 26, 20 and 8 mM, respectively. The activity constant, F s , decreased to 2 ml/h per g. These changes in kinetic constants were consistent with the hypothesis that anoxia accelerated sugar transport by releasing free carrier that was previously sequestered on the inside of the cell membrane.

Activation of particulate starch synthetase from Zea mays embryo

The effect of spermine on particulate ADP-glucose: starch synthetase from the developing embryo of sweet corn has been studied. Spermine induces a considerable increase of glucose incorporation from ADP-glucose into the starch granules. The change in kinetic constants, the distribution of incorporated glucose between amylose and amylopectin and the pattern of incorporation into starch granules or malto-oligosaccharides has been studied. The data were compared with those obtained with citrate ions.

Effect of polyhydric alcohols on kinetic parameters of enzymes

The effect of glycerol and other polyhydric compounds on the kinetic parameters of sixteen enzymes has been investigated. Substantial changes in K m and turnover rate were observed. Kinetic values obtained depended on pH as well as on the specific polyhydric compound added and its concentration. No pattern in direction or extent of change in kinetic constants was evident.

Glucose 6-Phosphate Dehydrogenase from Leuconostoc mesenteroides: KINETIC STUDIES

Glucose 6-phosphate dehydrogenase from Leuconostoc mesenteroides catalyzes the oxidation of glucose 6-phosphate by either NADP+ or NAD+. Steady state initial velocity studies and product inhibition studies using NADPH were conducted for the NADP-linked reaction catalyzed by this enzyme. The data were consistent with a simple, ordered, sequential mechanism for this reaction in which NADP+ is bound first to the enzyme and NADPH released last. Kinetic studies of the NAD-linked reaction indicated a more complex mechanism. Initial velocity studies, product, and alternate product inhibitions using NADH and NADPH, respectively, and alternate substrate studies using either NAD+ and NADP+ with glucose 6-phosphate, or glucose 6-phosphate and 2-deoxyglucose 6-phosphate with NAD+, suggested an ordered, sequential mechanism with isomerization of free enzyme. The enzyme form which binds NADH is proposed to be the same form binding NADP+ and NADPH; NAD+ is assumed to bind to an isomeric form. A detailed study of the effect of varying pH from 5.74 to 9.90 was carried out for the NAD-linked reaction. From the changes in kinetic constants with pH some tentative suggestions emerged concerning enzyme groups which may participate in the binding of NAD+ and glucose 6-phosphate. Among these, the most clear-cut was the participation of a group on the enzyme with pK' = 8.9 in binding of glucose 6-phosphate. Evidence was obtained that this group is not a cysteine. Further evidence, using pyridoxal 5'-phosphate, suggested that the group may be the e-NH2 group of a lysine.