Fast or Slow,Either Head Can Start the Processive Run of Kinesin-2 KIF3AC

Mammalian KIF3AC contains two distinct motor polypeptides and is best known for its role in organelle transport in neurons. Our recent studies showed that KIF3AC is as processive as conventional kinesin-1, suggesting that their ATPase mechanochemistry may be similar. However, the presence of two different motor polypeptides in KIF3AC implies that there must be a cellular advantage for the KIF3AC heterodimer. The hypothesis tested was whether there is an intrinsic bias within KIF3AC such that either KIF3A or KIF3C initiates the processive run. To pursue these experiments, a mechanistic approach was used to compare the pre-steady-state kinetics of KIF3AC to the kinetics of homodimeric KIF3AA and KIF3CC. The results indicate that microtubule collision at 11.4 μm⁻¹ s⁻¹ coupled with ADP release at 78 s⁻¹ are fast steps for homodimeric KIF3AA. In contrast, KIF3CC exhibits much slower microtubule association at 2.1 μm⁻¹ s⁻¹ and ADP release at 8 s⁻¹. For KIF3AC, microtubule association at 6.6 μm⁻¹ s⁻¹ and ADP release at 51 s⁻¹ are intermediate between the constants for KIF3AA and KIF3CC. These results indicate that either KIF3A or KIF3C can initiate the processive run. Surprisingly, the kinetics of the initial event of microtubule collision followed by ADP release for KIF3AC is not equivalent to 1:1 mixtures of KIF3AA plus KIF3CC homodimers at the same motor concentration. These results reveal that the intermolecular communication within the KIF3AC heterodimer modulates entry into the processive run regardless of whether the run is initiated by the KIF3A or KIF3C motor domain.     

Vanadate inhibits blue light-stimulated swelling of vicia guard cell protoplasts

When supplied under low chloride concentrations, vanadate inhibits the blue light-stimulated swelling of Vicia faba L. guard cell protoplasts in a dose-dependent fashion. The volume of guard cell protoplasts incubated in 10 mm K-imino-diacetic acid, 0.4 m mannitol, and 1 mm CaCl₂ remained essentially constant under 1000 μmol m⁻² s⁻¹ red light, but increased an average of 27% after 8 min of the addition of 50 μmol m⁻² s⁻¹ blue light to the background red light. At 500 μm, vanadate completely inhibits the response to blue light. Vanadate also inhibits the swelling of guard cell protoplasts stimulated by the H⁺-ATPase agonist fusicoccin. The vanadate sensitivity of the blue light-stimulated swelling implicates a proton-pumping ATPase as a component of the sensory transduction of blue light in guard cells.     

Nitric-oxide Dioxygenase Function of Human Cytoglobin with Cellular Reductants and in Rat Hepatocytes

Cytoglobin (Cygb) was investigated for its capacity to function as a NO dioxygenase (NOD) in vitro and in hepatocytes. Ascorbate and cytochrome b₅ were found to support a high NOD activity. Cygb-NOD activity shows respective K_m values for ascorbate, cytochrome b₅, NO, and O₂ of 0.25 mm, 0.3 μm, 40 nm, and ∼20 μm and achieves a k_cat of 0.5 s⁻¹. Ascorbate and cytochrome b₅ reduce the oxidized Cygb-NOD intermediate with apparent second order rate constants of 1000 m⁻¹ s⁻¹ and 3 × 10⁶ m⁻¹ s⁻¹, respectively. In rat hepatocytes engineered to express human Cygb, Cygb-NOD activity shows a similar k_cat of 1.2 s⁻¹, a K_m(NO) of 40 nm, and a k_cat/K_m(NO) (k′_NOD) value of 3 × 10⁷ m⁻¹ s⁻¹, demonstrating the efficiency of catalysis. NO inhibits the activity at [NO]/[O₂] ratios >1:500 and limits catalytic turnover. The activity is competitively inhibited by CO, is slowly inactivated by cyanide, and is distinct from the microsomal NOD activity. Cygb-NOD provides protection to the NO-sensitive aconitase. The results define the NOD function of Cygb and demonstrate roles for ascorbate and cytochrome b₅ as reductants.     

Human DNA Polymerase ν Catalyzes Correct and Incorrect DNA Synthesis with High Catalytic Efficiency

DNA polymerase ν (pol ν) is a low fidelity A-family polymerase with a putative role in interstrand cross-link repair and homologous recombination. We carried out pre-steady-state kinetic analysis to elucidate the kinetic mechanism of this enzyme. We found that the mechanism consists of seven steps, similar that of other A-family polymerases. pol ν binds to DNA with a K_d for DNA of 9.2 nm, with an off-rate constant of 0.013 s⁻¹and an on-rate constant of 14 μm⁻¹ s⁻¹. dNTP binding is rapid with K_d values of 20 and 476 μm for the correct and incorrect dNTP, respectively. Pyrophosphorylation occurs with a K_d value for PP_i of 3.7 mm and a maximal rate constant of 11 s⁻¹. Pre-steady-state kinetics, examination of the elemental effect using dNTPαS, and pulse-chase experiments indicate that a rapid phosphodiester bond formation step is flanked by slow conformational changes for both correct and incorrect base pair formation. These experiments in combination with computer simulations indicate that the first conformational change occurs with rate constants of 75 and 20 s⁻¹; rapid phosphodiester bond formation occurs with a K_eq of 2.2 and 1.7, and the second conformational change occurs with rate constants of 2.1 and 0.5 s⁻¹, for correct and incorrect base pair formation, respectively. The presence of a mispair does not induce the polymerase to adopt a low catalytic conformation. pol ν catalyzes both correct and mispair formation with high catalytic efficiency.     

Inability of tributyltin-induced chloride/hydroxyl exchange to stimulate calcium transport in mitochondria isolated from flight muscle of the sheep blowfly Lucilia cuprina

Tributyltin in the concentration range 1–4μm failed to stimulate Ca²⁺ transport by Lucilia flight-muscle mitochondria in a medium containing KCl and respiratory substrate but devoid of P_i, despite its promotion of a rapid Cl⁻/OH⁻ exchange. When 2mm-P_i was present, concentrations of tributyltin greater than 1μm inhibited the initial rate of Ca²⁺ transport and induced efflux of the ion from the mitochondria in Cl⁻- or NO₃⁻-containing media. Lower concentrations had little effect. Oligomycin added at up to 10μg/mg of mitochondrial protein had no effect on Ca²⁺ transport. By contrast, approx. 0.3μm-tributyltin completely inhibited respiration supported by α-glycerophosphate in either the presence or absence of added ADP. The data suggest that tributyltin can inhibit Ca²⁺ transport in Lucilia flight-muscle mitochondria other than by facilitating a Cl⁻/OH⁻ exchange or producing an oligomycin-like effect.     

Control of logarithmic growth rates of tobacco callus tissue by cytokinins

The growth rates of tobacco callus tissues on media containing 10⁻⁶ to 10 μm 6-(γ,γ-dimethylallylamino)purine (2iP) were measured. At concentrations of 10⁻⁴ μm and above growth rates were exponential and dependent on cytokinin concentration. At 2iP concentrations of 10⁻⁴ to 0.33 μm, the exponential rate was maintained for 4 to 5 doublings of fresh and dry weight. After this period a linear phase, resulting in approximately 1 doubling of weight, occurred. The growth of tissues on media containing higher than 0.33 μm 2iP was exponential for only about 15 days. At the end of this time, and well before they achieved half their final weight, they exhibited growth which was less rapid than logarithmic but more rapid than linear. Comparisons with zeatin, 6-benzylaminopurine and kinetin indicated that, although the maximum growth rates obtained with relatively high concentrations (0.1-1 μm) were similar, the naturally occurring cytokinins, 2iP and zeatin, promoted faster rates at lower concentrations (10⁻³-10⁻² μm) than did 6-benzylaminopurine and kinetin.     

Calcium Regulates Molecular Interactions of Otoferlin with Soluble NSF Attachment Protein Receptor (SNARE) Proteins Required for Hair Cell Exocytosis

Mutations in otoferlin, a C2 domain-containing ferlin family protein, cause non-syndromic hearing loss in humans (DFNB9 deafness). Furthermore, transmitter secretion of cochlear inner hair cells is compromised in mice lacking otoferlin. In the present study, we show that the C2F domain of otoferlin directly binds calcium (K_D = 267 μm) with diminished binding in a pachanga (D1767G) C2F mouse mutation. Calcium was found to differentially regulate binding of otoferlin C2 domains to target SNARE (t-SNARE) proteins and phospholipids. C2D–F domains interact with the syntaxin-1 t-SNARE motif with maximum binding within the range of 20–50 μm Ca²⁺. At 20 μm Ca²⁺, the dissociation rate was substantially lower, indicating increased binding (K_D = ∼10⁻⁹) compared with 0 μm Ca²⁺ (K_D = ∼10⁻⁸), suggesting a calcium-mediated stabilization of the C2 domain·t-SNARE complex. C2A and C2B interactions with t-SNAREs were insensitive to calcium. The C2F domain directly binds the t-SNARE SNAP-25 maximally at 100 μm and with reduction at 0 μm Ca²⁺, a pattern repeated for C2F domain interactions with phosphatidylinositol 4,5-bisphosphate. In contrast, C2F did not bind the vesicle SNARE protein synaptobrevin-1 (VAMP-1). Moreover, an antibody targeting otoferlin immunoprecipitated syntaxin-1 and SNAP-25 but not synaptobrevin-1. As opposed to an increase in binding with increased calcium, interactions between otoferlin C2F domain and intramolecular C2 domains occurred in the absence of calcium, consistent with intra-C2 domain interactions forming a “closed” tertiary structure at low calcium that “opens” as calcium increases. These results suggest a direct role for otoferlin in exocytosis and modulation of calcium-dependent membrane fusion.     

Partial Purification of the NADH-Nitrate Reductase Complex from Chlorella pyrenoidosa

The nitrate reductase complex from Chlorella pyrenoidosa has been purified by a procedure which includes as main steps, ammonium sulfate fractionation, polyethylene glycol treatment, and DEAE-cellulose chromatography. The Michaelis constants for NADH, FAD, and NO₃⁻ in the NADH-nitrate reductase assay are 10 μm, 2.6 μm, and 0.23 mm, respectively. Heat treatment exerts varying effects on the enzymatic activities associated with the nitrate reductase complex.     

Basic Properties of Rotary Dynamics of the Molecular Motor Enterococcus hirae V1-ATPase

V-ATPases are rotary molecular motors that generally function as proton pumps. We recently solved the crystal structures of the V₁ moiety of Enterococcus hirae V-ATPase (EhV₁) and proposed a model for its rotation mechanism. Here, we characterized the rotary dynamics of EhV₁ using single-molecule analysis employing a load-free probe. EhV₁ rotated in a counterclockwise direction, exhibiting two distinct rotational states, namely clear and unclear, suggesting unstable interactions between the rotor and stator. The clear state was analyzed in detail to obtain kinetic parameters. The rotation rates obeyed Michaelis-Menten kinetics with a maximal rotation rate (V_max) of 107 revolutions/s and a Michaelis constant (K_m) of 154 μm at 26 °C. At all ATP concentrations tested, EhV₁ showed only three pauses separated by 120°/turn, and no substeps were resolved, as was the case with Thermus thermophilus V₁-ATPase (TtV₁). At 10 μm ATP (⪡K_m), the distribution of the durations of the ATP-waiting pause fit well with a single-exponential decay function. The second-order binding rate constant for ATP was 2.3 × 10⁶ m⁻¹ s⁻¹. At 40 mm ATP (⪢K_m), the distribution of the durations of the catalytic pause was reproduced by a consecutive reaction with two time constants of 2.6 and 0.5 ms. These kinetic parameters were similar to those of TtV₁. Our results identify the common properties of rotary catalysis of V₁-ATPases that are distinct from those of F₁-ATPases and will further our understanding of the general mechanisms of rotary molecular motors.     

Alginate Lyases from Alginate-Degrading Vibrio splendidus 12B01 Are Endolytic

Alginate lyases are enzymes that degrade alginate through β-elimination of the glycosidic bond into smaller oligomers. We investigated the alginate lyases from Vibrio splendidus 12B01, a marine bacterioplankton species that can grow on alginate as its sole carbon source. We identified, purified, and characterized four polysaccharide lyase family 7 alginates lyases, AlyA, AlyB, AlyD, and AlyE, from V. splendidus 12B01. The four lyases were found to have optimal activity between pH 7.5 and 8.5 and at 20 to 25°C, consistent with their use in a marine environment. AlyA, AlyB, AlyD, and AlyE were found to exhibit a turnover number (k_cat) for alginate of 0.60 ± 0.02 s⁻¹, 3.7 ± 0.3 s⁻¹, 4.5 ± 0.5 s⁻¹, and 7.1 ± 0.2 s⁻¹, respectively. The K_m values of AlyA, AlyB, AlyD, and AlyE toward alginate were 36 ± 7 μM, 22 ± 5 μM, 60 ± 2 μM, and 123 ± 6 μM, respectively. AlyA and AlyB were found principally to cleave the β-1,4 bonds between β-d-mannuronate and α-l-guluronate and subunits; AlyD and AlyE were found to principally cleave the α-1,4 bonds involving α-l-guluronate subunits. The four alginate lyases degrade alginate into longer chains of oligomers.     

Effectors of rat-heart hexokinases and the control of rates of glucose phosphorylation in the perfused rat heart

1. The kinetic properties of the soluble and particulate hexokinases from rat heart have been investigated. 2. For both forms of the enzyme, the K_m for glucose was 45μm and the K_m for ATP 0·5mm. Glucose 6-phosphate was a non-competitive inhibitor with respect to glucose (K_i 0·16mm for the soluble and 0·33mm for the particulate enzyme) and a mixed inhibitor with respect to ATP (K_i 80μm for the soluble and 40μm for the particulate enzyme). ADP and AMP were competitive inhibitors with respect to ATP (K_i for ADP was 0·68mm for the soluble and 0·60mm for the particulate enzyme; K_i for AMP was 0·37mm for the soluble and 0·16mm for the particulate enzyme). P_i reversed glucose 6-phosphate inhibition with both forms at 10mm but not at 2mm, with glucose 6-phosphate concentrations of 0·3mm or less for the soluble and 1mm or less for the particulate enzyme. 3. The total activity of hexokinase in normal hearts and in hearts from alloxan-diabetic rats was 21·5μmoles of glucose phosphorylated/min./g. dry wt. of ventricle at 25°. The temperature coefficient Q₁₀ between 22° and 38·5° was 1·93; the ratio of the soluble to the particulate enzyme was 3:7. 4. The kinetic data have been used to predict rates of glucose phosphorylation in the perfused heart at saturating concentrations of glucose from measured concentrations of ATP, glucose 6-phosphate, ADP and AMP. These have been compared with the rates of glucose phosphorylation measured with precision in a small-volume recirculation perfusion apparatus, which is described. The correlation between predicted and measured rates was highly significant and their ratio was 1·07. 5. These findings are consistent with the control of glucose phosphorylation in the perfused heart by glucose 6-phosphate concentration, subject to certain assumptions that are discussed in detail.     

Effect of l-Canavanine on Nitrate Reductase in Corn Roots

l-Canavanine inhibits the appearance of nitrate reductase (NADH-nitrate oxidoreductase, EC 1.6.6.1) in both root tips and mature root sections of corn (Zea mays L.). Ten-fold more canavanine was required to cause a 50% reduction in the level of nitrate reductase activity (NRA) in root tips than in mature root sections. For example with one particular batch of seeds 500 μm canavanine was effective in root tips whereas only 50 μm was required in mature root sections. In root tips arginine (1 mm) completely reversed the effect of 1 mm canavanine. In mature root sections higher concentrations of arginine (approximately 5 mm) were required for a complete reversal of the canavanine effect. Additions of canavanine to roots after a period of 3 hours with 5 mm KNO₃ resulted in a loss of NRA. NO₃⁻ protected nitrate reductase from this inactivation in both root tip and mature root sections.     

Identification,Purification, and Characterization of a Novel Amino Acid Racemase,Isoleucine 2-Epimerase,from Lactobacillus Species

Accumulation of d-leucine, d-allo-isoleucine, and d-valine was observed in the growth medium of a lactic acid bacterium, Lactobacillus otakiensis JCM 15040, and the racemase responsible was purified from the cells and identified. The N-terminal amino acid sequence of the purified enzyme was GKLDKASKLI, which is consistent with that of a putative γ-aminobutyrate aminotransferase from Lactobacillus buchneri. The putative γ-aminobutyrate aminotransferase gene from L. buchneri JCM 1115 was expressed in recombinant Escherichia coli and then purified to homogeneity. The enzyme catalyzed the racemization of a broad spectrum of nonpolar amino acids. In particular, it catalyzed at high rates the epimerization of l-isoleucine to d-allo-isoleucine and d-allo-isoleucine to l-isoleucine. In contrast, the enzyme showed no γ-aminobutyrate aminotransferase activity. The relative molecular masses of the subunit and native enzyme were estimated to be about 49 kDa and 200 kDa, respectively, indicating that the enzyme was composed of four subunits of equal molecular masses. The K_m and V_max values of the enzyme for l-isoleucine were 5.00 mM and 153 μmol·min⁻¹·mg⁻¹, respectively, and those for d-allo-isoleucine were 13.2 mM and 286 μmol·min⁻¹·mg⁻¹, respectively. Hydroxylamine and other inhibitors of pyridoxal 5′-phosphate-dependent enzymes completely blocked the enzyme activity, indicating the enzyme requires pyridoxal 5′-phosphate as a coenzyme. This is the first evidence of an amino acid racemase that specifically catalyzes racemization of nonpolar amino acids at the C-2 position.     

myo-Inositol homeostasis in foetal rabbit lung

In several species, lung maturation is accompanied by a decline in the phosphatidylinositol content of lung surfactant and a concomitant increase in its phosphatidylglycerol content. To examine the possibility that this developmental change is influenced by the availability of myo-inositol, potential sources of myo-inositol for the developing rabbit lung were investigated. On day 28 of gestation the myo-inositol content of foetal rabbit lung tissue (2.3±0.5μmol/g of tissue) was not significantly different from that of adult lung tissue but the activity of d-glucose 6-phosphate:1l-myo-inositol 1-phosphate cyclase (cyclase) in foetal lung tissue (81.0±9.0nmol·h⁻¹·g of tissue⁻¹) was higher than that found in adult lung tissue (23.2±1.0nmol·h⁻¹·g of tissue⁻¹). Day 28 foetal rabbit lung tissue was found also to take up myo-inositol by a specific, energy-dependent, Na⁺-requiring mechanism. Half-maximal uptake of myo-inositol by foetal rabbit lung slices was observed when the concentration of myo-inositol in the incubation medium was 85μm. When the myo-inositol concentration was 1mm (but not 100μm) the addition of glucose (5.5mm) stimulated myo-inositol uptake. myo-Inositol uptake was observed also in adult rabbit lung and was found to be sub-maximal at the concentration of myo-inositol found in adult rabbit serum. The concentration of myo-inositol in the serum of pregnant adult rabbits (47.5±5.5μm) was significantly lower than that of non-pregnant adult female rabbits (77.9±9.2μm). On day 28 of gestation the concentration of myo-inositol in foetal serum (175.1±12.0μm) was much less than on day 25, but more than that found on day 30. A transient post-partum increase in the concentration of myo-inositol in serum was followed by a rapid decline. Much of the myo-inositol in foetal rabbit serum probably originates from the placenta, where on day 28 of gestation a high cyclase activity (527±64nmol·h⁻¹·g of tissue⁻¹) was measured. The gestational decline in serum myo-inositol concentration, together with the decreasing cyclase activity of the lungs, is consistent with the view that maturation of the lungs is accompanied by decreased availability of myo-inositol to this tissue.     

Substrate Control of Internal Electron Transfer in Bacterial Nitric-oxide Reductase

Nitric -oxide reductase (NOR) from Paracoccus denitrificans catalyzes the reduction of nitric oxide (NO) to nitrous oxide (N₂O) (2NO + 2H⁺ + 2e⁻ →N₂O + H₂O) by a poorly understood mechanism. NOR contains two low spin hemes c and b, one high spin heme b₃, and a non-heme iron Fe_B. Here, we have studied the reaction between fully reduced NOR and NO using the “flow-flash” technique. Fully (four-electron) reduced NOR is capable of two turnovers with NO. Initial binding of NO to reduced heme b₃ occurs with a time constant of ∼1 μs at 1.5 mm NO, in agreement with earlier studies. This reaction is [NO]-dependent, ruling out an obligatory binding of NO to Fe_B before ligation to heme b₃. Oxidation of hemes b and c occurs in a biphasic reaction with rate constants of 50 s⁻¹ and 3 s⁻¹ at 1.5 mm NO and pH 7.5. Interestingly, this oxidation is accelerated as [NO] is lowered; the rate constants are 120 s⁻¹ and 12 s⁻¹ at 75 μm NO. Protons are taken up from solution concomitantly with oxidation of the low spin hemes, leading to an acceleration at low pH. This effect is, however, counteracted by a larger degree of substrate inhibition at low pH. Our data thus show that substrate inhibition in NOR, previously observed during multiple turnovers, already occurs during a single oxidative cycle. Thus, NO must bind to its inhibitory site before electrons redistribute to the active site. The further implications of our data for the mechanism of NO reduction by NOR are discussed.     

The reversibility of adenosine triphosphate cleavage by myosin

For the simplest kinetic model the reverse rate constants (k₋₁ and k₋₂) associated with ATP binding and cleavage on purified heavy meromyosin and heavy meromyosin subfragment 1 from rabbit skeletal muscle in the presence of 5mm-MgCl₂, 50mm-KCl and 20mm-Tris–HCl buffer at pH8.0 and 22°C are: k₋₁<0.02s⁻¹ and k₋₁=16s⁻¹. Apparently, higher values of k₋₁ and k₋₂ are found with less-purified protein preparations. The values of k₋₁ and k₋₂ satisfy conditions required by previous ¹⁸O-incorporation studies of H₂¹⁸O into the P_i moiety on ATP hydrolysis and suggest that the cleavage step does involve hydrolysis of ATP or formation of an adduct between ATP and water. The equilibrium constant for the cleavage step at the myosin active site is 9. If the cycle of events during muscle contraction is described by the model proposed by Lymn & Taylor (1971), the fact that there is only a small negative standard free-energy change for the cleavage step is advantageous for efficient chemical to mechanical energy exchange during muscle contraction.     

Inhibition of purine phosphoribosyltransferases of Ehrlich ascites-tumour cells by 6-mercaptopurine

1. The formation of adenosine 5′-phosphate, guanosine 5′-phosphate and inosine 5′-phosphate from [8-¹⁴C]adenine, [8-¹⁴C]guanine and [8-¹⁴C]hypoxanthine respectively in the presence of 5-phosphoribosyl pyrophosphate and an extract from Ehrlich ascites-tumour cells was assayed by a method involving liquid-scintillation counting of the radioactive nucleotides on diethylaminoethylcellulose paper. The results obtained with guanine were confirmed by a spectrophotometric assay which was also used to assay the conversion of 6-mercaptopurine and 5-phosphoribosyl pyrophosphate into 6-thioinosine 5′-phosphate in the presence of 6-mercaptopurine phosphoribosyltransferase from these cells. 2. At pH 7·8 and 25° the Michaelis constants for adenine, guanine and hypoxanthine were 0·9 μm, 2·9 μm and 11·0 μm in the assay with radioactive purines; the Michaelis constant for guanine in the spectrophotometric assay was 2·6 μm. At pH 7·9 the Michaelis constant for 6-mercaptopurine was 10·9 μm. 3. 25 μm-6-Mercaptopurine did not inhibit adenine phosphoribosyltransferase. 6-Mercaptopurine is a competitive inhibitor of guanine phosphoribosyltransferase (K_i 4·7 μm) and hypoxanthine phosphoribosyltransferase (K_i 8·3 μm). Hypoxanthine is a competitive inhibitor of guanine phosphoribosyltransferase (K_i 3·4 μm). 4. Differences in kinetic parameters and in the distribution of phosphoribosyltransferase activities after electrophoresis in starch gel indicate that different enzymes are involved in the conversion of adenine, guanine and hypoxanthine into their nucleotides. 5. From the low values of K_i for 6-mercaptopurine, and from published evidence that ascites-tumour cells require supplies of purines from the host tissues, it is likely that inhibition of hypoxanthine and guanine phosphoribosyltransferases by free 6-mercaptopurine is involved in the biological activity of this drug.     

The activities and kinetic properties of purine phosphoribosyltransferases in developing mouse liver

1. The total activity of adenine phosphoribosyltransferase/liver of mice remained constant from 1 to 16 days after birth despite a fourfold increase in liver weight. The total activity of this enzyme increased fivefold from 16 to 36 days and then remained relatively constant at least until 96 days after birth. Total hypoxanthine-phosphoribosyltransferase activity/liver steadily increased between 1 and 57 days after birth. 2. The mean K_m of 5-phosphoribosyl pyrophosphate with adenine phosphoribosyltransferase was 10·1μm between 3 and 11 days, at 64 days and at 96 days after birth. Between 17 and 51 days the mean K_m value was 3·0μm. The K_m of 5-phosphoribosyl pyrophosphate with hypoxanthine phosphoribosyltransferase remained constant at 28·2μm between 2 and 64 days. 3. Adenine-phosphoribosyltransferase activity was stimulated between 15 and 83% by 60μm-ATP when extracts were made between 3 and 11 days, at 64 days or at 96 days after birth. Between 17 and 51 days ATP had little stimulatory effect on the activity of this enzyme. 4. AMP competed with 5-phosphoribosyl pyrophosphate in the reaction catalysed by adenine phosphoribosyltransferase. Liver extracts containing enzyme with a low value of K_m for 5-phosphoribosyl pyrophosphate (3μm) had a K_m/K_i ratio approximately half that of extracts with a high value of K_m (10μm). 5. The results indicate that two different forms of adenine phosphoribosyltransferase can exist in mouse liver at different stages of development. The physiological significance of these findings is discussed.     

Detection of Reaction Intermediates during Human Cystathionine β-Synthase-monitored Turnover and H2S Production

Human cystathionine β-synthase (CBS), a novel heme-containing pyridoxal 5′-phosphate enzyme, catalyzes the condensation of homocysteine and serine or cysteine to produce cystathionine and H₂O or H₂S, respectively. The presence of heme in CBS has limited spectrophotometric characterization of reaction intermediates by masking the absorption of the pyridoxal 5′-phosphate cofactor. In this study, we employed difference stopped-flow spectroscopy to characterize reaction intermediates formed under catalytic turnover conditions. The reactions of l-serine and l-cysteine with CBS resulted in the formation of a common aminoacrylate intermediate (k_obs = 0.96 ± 0.02 and 0.38 ± 0.01 mm⁻¹ s⁻¹, respectively, at 24 °C) with concomitant loss of H₂O and H₂S and without detectable accumulation of the external aldimine or other intermediates. Homocysteine reacted with the aminoacrylate intermediate with k_obs = 40.6 ± 3.8 s⁻¹ and re-formed the internal aldimine. In the reverse direction, CBS reacted with cystathionine, forming the aminoacrylate intermediate with k_obs = 0.38 ± 0.01 mm⁻¹ s⁻¹. This study provides the first insights into the pre-steady-state kinetic mechanism of human CBS and indicates that the reaction is likely to be limited by a conformational change leading to product release.