Substrate binding is the rate-limiting step in thromboxane synthase catalysis

Thromboxane synthase (TXAS) is a "non-classical" cytochrome P450. Without any need for an external electron donor, or for a reductase or molecular oxygen, it uses prostaglandin H2 (PGH2) to catalyze either an isomerization reaction to form thromboxane A2 (TXA2) or a fragmentation reaction to form 12-l-hydroxy-5,8,10-heptadecatrienoic acid and malondialdehyde (MDA) at a ratio of 1:1:1 (TXA2:heptadecatrienoic acid:MDA). We report here kinetics of TXAS with heme ligands in binding study and with PGH2 in enzymatic study. We determined that 1) binding of U44069, an oxygen-based ligand, is a two-step process; U44069 first binds TXAS, then ligates the heme-iron with a maximal rate constant of 105-130 s(-1); 2) binding of cyanide, a carbon-based ligand, is a one-step process with k(on) of 2.4 M(-1) s(-1) and k(off) of 0.112 s(-1); and 3) both imidazole and clotrimazole (nitrogen-based ligands) bind TXAS in a two-step process; an initial binding to the heme-iron with on-rate constants of 8.4 x 10(4) M(-1) s(-1) and 1.5 x 10(5) M(-1) s(-1) for imidazole and clotrimazole, respectively, followed by a slow conformational change with off-rate constants of 8.8 s(-1) and 0.53 s(-1), respectively. The results of our binding study indicate that the TXAS active site is hydrophobic and spacious. In addition, steady-state kinetic study revealed that TXAS consumed PGH2 at a rate of 3,800 min(-1) and that the k(cat)/K(m) for PGH2 consumption was 3 x 10(6) M(-1) s(-1). Based on these data, TXAS appears to be a very efficient catalyst. Surprisingly, rapid-scan stopped-flow experiments revealed marginal absorbance changes upon mixing TXAS with PGH2, indicating minimal accumulation of any heme-derived intermediates. Freeze-quench EPR measurements for the same reaction showed minimal change of heme redox state. Further kinetic analysis using a combination of rapid-mixing chemical quench and computer simulation showed that the kinetic parameters of TXAS-catalyzed reaction are: PGH2 bound TXAS at a rate of 1.2-2.0 x 10(7) M(-1) s(-1); the rate of catalytic conversion of PGH2 to TXA2 or MDA was at least 15,000 s(-1) and the lower limit of the rates for products release was 4,000-6,000 s(-1). Given that the cellular PGH2 concentration is quite low, we concluded that under physiological conditions, the substrate-binding step is the rate-limiting step of the TXAS-catalyzed reaction, in sharp contrast with "classical" P450 enzymes.     

Dissociation from the oligomeric state is the rate-limiting step in fibril formation by kappa-casein

Amyloid fibrils are aggregated and precipitated forms of protein in which the protein exists in highly ordered, long, unbranching threadlike formations that are stable and resistant to degradation by proteases. Fibril formation is an ordered process that typically involves the unfolding of a protein to partially folded states that subsequently interact and aggregate through a nucleation-dependent mechanism. Here we report on studies investigating the molecular basis of the inherent propensity of the milk protein, kappa-casein, to form amyloid fibrils. Using reduced and carboxymethylated kappa-casein (RCMkappa-CN), we show that fibril formation is accompanied by a characteristic increase in thioflavin T fluorescence intensity, solution turbidity, and beta-sheet content of the protein. However, the lag phase of RCMkappa-CN fibril formation is independent of protein concentration, and the rate of fibril formation does not increase upon the addition of seeds (preformed fibrils). Therefore, its mechanism of fibril formation differs from the archetypal nucleation-dependent aggregation mechanism. By digestion with trypsin or proteinase K and identification by mass spectrometry, we have determined that the region from Tyr(25) to Lys(86) is incorporated into the core of the fibrils. We suggest that this region, which is predicted to be aggregation-prone, accounts for the amyloidogenic nature of kappa-casein. Based on these data, we propose that fibril formation by RCMkappa-CN occurs through a novel mechanism whereby the rate-limiting step is the dissociation of an amyloidogenic precursor from an oligomeric state rather than the formation of stable nuclei, as has been described for most other fibril-forming systems.     

Oxidative dimer formation is the critical rate-limiting step for Parkinson's disease alpha-synuclein fibrillogenesis

Intraneuronal deposition of alpha-synuclein as fibrils and oxidative stress are both implicated in the pathogenesis of Parkinson's disease. We found that the critical rate-limiting step in nucleation of alpha-synuclein fibrils under physiological conditions is the oxidative formation and accumulation of a dimeric, dityrosine cross-linked prenucleus. Dimer formation is accelerated for the pathogenic A30P and A53T mutant alpha-synucleins, because of their greater propensity to self-interact, which is reflected in the smaller values of the osmotic second virial coefficient compared to that of wild-type synuclein. Our finding that oxidation is an essential step in alpha-synuclein aggregation supports a mechanism of Parkinson's disease pathogenesis in which the separately studied pathogenic factors of oxidative stress and alpha-synuclein aggregation converge at the critical step of alpha-synuclein dimer formation.     

Processing of VSVG protein is not a rate-limiting step for its efflux from the Golgi complex

The secretory membrane system is comprised of membrane-bound organelles defined by specific sets of proteins that function in sequential modification of cargo proteins and lipids. This processing of cargo proteins and lipids is coupled to their secretory transport. Here, we investigated the effect of inhibiting N-glycan processing by swainsonine, an inhibitor of Golgi alpha1,2-mannosidase-II, on secretory transport of the thermo-reversible tsO45 mutant of vesicular stomatitis virus glycoprotein tagged with green fluorescent protein (VSVG-FP). Quantitative analysis using kinetic modeling combined with live cell imaging was used to derive the rate coefficients that delineate secretory transport of VSVG-FP. We found that neither inhibition of N-glycan processing nor elimination by mutagenesis of the first of the two asparagine-linked glycans had any significant effect on the rate of VSVG-FP transport through the Golgi. These data suggest that at least for VSVG, the multi-enzymatic process of N-glycan modification does not comprise a rate-limiting step for its Golgi efflux.     

Probing a rate-limiting step by mutational perturbation of AdoMet binding in the HhaI methyltransferase

DNA methylation plays important roles via regulation of numerous cellular mechanisms in diverse organisms, including humans. The paradigm bacterial methyltransferase (MTase) HhaI (M.HhaI) catalyzes the transfer of a methyl group from the cofactor S-adenosyl-L-methionine (AdoMet) onto the target cytosine in DNA, yielding 5-methylcytosine and S-adenosyl-L-homocysteine (AdoHcy). The turnover rate (k cat) of M.HhaI, and the other two cytosine-5 MTases examined, is limited by a step subsequent to methyl transfer; however, no such step has so far been identified. To elucidate the role of cofactor interactions during catalysis, eight mutants of Trp41, which is located in the cofactor binding pocket, were constructed and characterized. The mutants show full proficiency in DNA binding and base-flipping, and little variation is observed in the apparent methyl transfer rate k chem as determined by rapid-quench experiments using immobilized fluorescent-labeled DNA. However, the Trp41 replacements with short side chains substantially perturb cofactor binding (100-fold higher K(AdoMet)D and K(AdoMet)M) leading to a faster turnover of the enzyme (10-fold higher k cat). Our analysis indicates that the rate-limiting breakdown of a long-lived ternary product complex is initiated by the dissociation of AdoHcy or the opening of the catalytic loop in the enzyme.     

Cytochrome b6f is a dimeric protochlorophyll a binding complex in etioplasts

The cytochrome b6f complex is a dimeric protein complex that is of central importance for photosynthesis to carry out light driven electron and proton transfer in chloroplasts. One molecule of chlorophyll a was found to associate per cytochrome b6f monomer and the structural or functional importance of this is discussed. We show that etioplasts which are devoid of chlorophyll a already contain dimeric cytochrome b6f. However, the phytylated chlorophyll precursor protochlorophyll a, and not chlorophyll a, is associated with subunit b6. The data imply that a phytylated tetrapyrrol is an essential structural requirement for assembly of cytochrome b6f.     

The rate-limiting step in the actomyosin adenosinetriphosphatase cycle

We have previously shown that myosin does not have to detach from actin during each cycle of ATP hydrolysis. In the present study, using the A-1 isoenzyme of myosin subfragment 1, we have investigated the nature of the rate-limiting steps in the ATPase cycle. Our results show that, at 15 degrees C, at very low ionic strength, KATPase determined from the double-reciprocal plot of ATPase activity vs. actin concentration is more than 6-fold stronger than KBINDING determined by directly measuring the binding of A-1 myosin subfragment 1 to actin during steady-state ATP hydrolysis. Computer modeling shows that this large difference between KATPase and KBINDING is not compatible with Pi release being the rate-limiting step in the ATPase cycle. If Pi release is not rate limiting, it is possible that the ATP hydrolysis step, itself, is rate limiting. However, this predicts that, at high actin concentration, the value of the initial Pi burst should be close to zero. Therefore, we measured the magnitude of the initial Pi burst in the presence of actin, using both direct measurement and measurement of relative fluorescence magnitude. Our results suggest that the magnitude of the initial Pi burst in the presence of actin is considerably higher than would be expected if the ATP hydrolysis step were the rate-limiting step in the ATPase cycle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Breaking non-native hydrophobic clusters is the rate-limiting step in the folding of an alanine-based peptide

The formation mechanism of an alanine-based peptide has been studied by all-atom molecular dynamics simulations with a recently developed all-atom point-charge force field and the Generalize Born continuum solvent model at an effective salt concentration of 0.2M. Thirty-two simulations were conducted. Each simulation was performed for 100 ns. A surprisingly complex folding process was observed. The development of the helical content can be divided into three phases with time constants of 0.06-0.08, 1.4-2.3, and 12-13 ns, respectively. Helices initiate extreme rapidly in the first phase similar to that estimated from explicit solvent simulations. Hydrophobic collapse also takes place in this phase. A folding intermediate state develops in the second phase and is unfolded to allow the peptide to reach the transition state in the third phase. The folding intermediate states are characterized by the two-turn short helices and the transition states are helix-turn-helix motifs-both of which are stabilized by hydrophobic clusters. The equilibrium helical content, calculated by both the main-chain Phi-Psi torsion angles and the main-chain hydrogen bonds, is 64-66%, which is in remarkable agreement with experiments. After corrected for the solvent viscosity effect, an extrapolated folding time of 16-20 ns is obtained that is in qualitative agreement with experiments. Contrary to the prevailing opinion, neither initiation nor growth of the helix is the rate-limiting step. Instead, the rate-limiting step for this peptide is breaking the non-native hydrophobic clusters in order to reach the transition state. The implication to the folding mechanisms of proteins is also discussed.     

A rate-limiting step of enteropeptidase hydrolysis

It has been shown for the first time that deacylation is the rate-limiting step in the enteropeptidase-catalyzed hydrolysis of highly effective oligopeptide substrates containing four Asp residues in positions P2–P5. On the other hand, the rate-limiting step in the hydrolysis of low-efficiency peptide substrates containing less than four Asp or Glu residues in positions P2–P5 is acylation, as it has previously been suggested for all amide and peptide substrates of serine proteases on the basis of classical works of Bender et al. The method of introduction of an additional nucleophile or another effector that selectively affects the deacylation step was used to determine the rate-limiting step in the enteropeptidase hydrolysis of N ^α-benzyloxycarbonyl-L-lysine thiobenzyl ester, the highly efficient amide substrate GlyAsp₄-Lys β-naphthyl amide, and the low-efficiency peptide substrate VLSAADK-GNVKAAWG (where a hyphen denotes the hydrolysis site).     

On the rate-limiting step in W-hydroxylation of lauric acid

Incubation of lauric acid with rat liver microsomes and NADPH yielded a mixture of 11-D-, 11-L-, and 12-hydroxylauric acids. 11-L- and 11-D-hydroxylations of 11-²H₂-lauric acid were accompanied by marked isotope effects, indicating that in these reactions splitting of the CH bond is rate limiting. When 11- and 12-hydroxylations of lauric acid were carried out in D₂O, 12-hydroxylation but not 11-hydroxylation was inhibited, showing that different steps are rate limiting in the two reactions.     

The dimeric SOS mutagenesis protein UmuD is active as a monomer

The homodimeric umuD gene products play key roles in regulating the cellular response to DNA damage in Escherichia coli. UmuD(2) is composed of 139-amino acid subunits and is up-regulated as part of the SOS response. Subsequently, damage-induced RecA·ssDNA nucleoprotein filaments mediate the slow self-cleavage of the N-terminal 24-amino acid arms yielding UmuD'(2). UmuD(2) and UmuD'(2) make a number of distinct protein-protein contacts that both prevent and facilitate mutagenic translesion synthesis. Wild-type UmuD(2) and UmuD'(2) form exceptionally tight dimers in solution; however, we show that the single amino acid change N41D generates stable, active UmuD and UmuD' monomers that functionally mimic the dimeric wild-type proteins. The UmuD N41D monomer is proficient for cleavage and interacts physically with DNA polymerase IV (DinB) and the β clamp. Furthermore, the N41D variants facilitate UV-induced mutagenesis and promote overall cell viability. Taken together, these observations show that a monomeric form of UmuD retains substantial function in vivo and in vitro.     

A desolvation barrier to hydrophobic cluster formation may contribute to the rate-limiting step in protein folding.

To gain insight into the free energy changes accompanying protein hydrophobic core formation, we have used computer simulations to study the formation of small clusters of nonpolar solutes in water. A barrier to association is observed at the largest solute separation that does not allow substantial solvent penetration. The barrier reflects an effective increase in the size of the cavity occupied by the expanded but water-excluding cluster relative to both the close-packed cluster and the fully solvated separated solutes; a similar effect may contribute to the barrier to protein folding/unfolding. Importantly for the simulation of protein folding without explicit solvent, we find that the interactions between nonpolar solutes of varying size and number can be approximated by a linear function of the molecular surface, but not the solvent-accessible surface of the solutes. Comparison of the free energy of cluster formation to that of dimer formation suggests that the assumption of pair additivity implicit in current protein database derived potentials may be in error.     

Methionine and alanine substitutions show that the formation of wild-type-like structure in the carboxy-terminal domain of T4 lysozyme is a rate-limiting step in folding.

In an attempt to identify a systematic relation between the structure of a protein and its folding kinetics, the rate of folding was determined for 20 mutants of T4 lysozyme in which a bulky, buried, nonpolar wild-type residue (Leu, Ile, Phe, Val, or Met) was substituted with alanine. Methionine, which approximated the size of the original side chain but which is of different shape and flexibility, was also substituted at most of the same sites. Mutations that substantially destabilize the protein and are located in the carboxy-terminal domain generally slow the rate of folding. Destabilizing mutations in the amino-terminal domain, however, have little effect on the rate of folding. Mutations that have little effect on stability tend to have little effect on the rate, no matter where they are located. These results suggest that, at the rate-limiting step, elements of structure in the C-terminal domain are formed and have a structure similar to that of the fully folded protein. Consistent with this, two variants that somewhat increase the rate of folding (Phe104 --> Met and Val149 --> Met) are located within the carboxy-terminal domain and maintain or improve packing with very little perturbation of the wild-type structure.     

Characteristics of carbohydrate degradation and the rate-limiting step in anaerobic digestion

The characteristics of the degradation of cellulose, soluble starch, and glucose in the acidogenic phase and the effects of the substrate loading rate and biological solids retention time on the methanogenic phase of anaerobic digestion were investigated. The results obtained from continuous experiments using laboratory-scale anaerobic chemostat reactors elucidated the true rate-limiting step of anaerobic digestion. The specific rate of substrate utilization decreased in the following order: glucose, soluble starch, acetic acid, and cellulose. The rate of the hydrolysis of cellulose was so low that this was shown to be the rate-limiting step in overall anaerobic digestion. Among methanogenic bacteria Methanosarcina would provide a higher substrate utilization rate than Methanothrix, and the maximum allowable substrate loading rate in the methanogenic phase was 11.2 g acetic acid/L day.