Effects on substrate profile by mutational substitutions at positions 164 and 179 of the class A TEM(pUC19) beta-lactamase from Escherichia coli.

We investigated the effects of mutations at positions 164 and 179 of the TEM(pUC19) beta-lactamase on turnover of substrates. The direct consequence of some mutations at these sites is that clinically important expanded-spectrum beta-lactams, such as third-generation cephalosporins, which are normally exceedingly poor substrates for class A beta-lactamases, bind the active site of these mutant enzymes more favorably. We employed site-saturation mutagenesis at both positions 164 and 179 to identify mutant variants of the parental enzyme that conferred resistance to expanded-spectrum beta-lactams by their enhanced ability to turn over these antibiotic substrates. Four of these mutant variants, Arg(164) --> Asn, Arg(164) --> Ser, Asp(179) --> Asn, and Asp(179) --> Gly, were purified and the details of their catalytic properties were examined in a series of biochemical and kinetic experiments. The effects on the kinetic parameters were such that either activity with the expanded-spectrum beta-lactams remained unchanged or, in some cases, the activity was enhanced. The affinity of the enzyme for these poorer substrates (as defined by the dissociation constant, K(s)) invariably increased. Computation of the microscopic rate constants (k(2) and k(3)) for turnover of these poorer substrates indicated either that the rate-limiting step in turnover was the deacylation step (governed by k(3)) or that neither the acylation nor deacylation became the sole rate-limiting step. In a few instances, the rate constants for both the acylation (k(2)) and deacylation (k(3)) of the extended-spectrum beta-lactamase were enhanced. These results were investigated further by molecular modeling experiments, using the crystal structure of the TEM(pUC19) beta-lactamase. Our results indicated that severe steric interactions between the large 7beta functionalities of the expanded-spectrum beta-lactams and the Omega-loop secondary structural element near the active site were at the root of the low affinity by the enzyme for these substrates. These conclusions were consistent with the proposal that the aforementioned mutations would enlarge the active site, and hence improve affinity.     

Kinetic investigation of the alpha-chymotrypsin-catalyzed hydrolysis of peptide-ester substrates. The relationship between the structure of the peptide moiety and reactivity.

A number of peptide-ester substrates of the general structure Ac-Lxn-...-Lx2-Lx1-OMe have been synthesized and their alpha-chymotrypsin-catalyzed hydrolysis studied. The kinetic analysis involved varying the concentration of substrate and methanol product, and measuring rates along the entire progression curve. For the dipeptide esters Ac-Lx2-Lx1-OMe and the amino-acid derivatives Ac-Lx1-OMe the following constants could be determined: the dissociation constant of the enzyme-substrate complex, KEA, both rate constants of the acylation step, k23 and k32, and the forward rate constant of the deacylation step, k31. For the tripeptide ester Ac-Ala-Ala-Tyr-OMe it appears that the rate constant for the dissociation of the enzyme-substrate complex, k21, is smaller than the rate constant for acylation, k23. Thus, for this substrate only the association and dissociation rate constants k12 and k21 could be determined and the values of k23, k32 and k31 only indirectly estimated. The influence of structural changes in the peptide moiety of the substrates on reactivity has been established by comparing the rate constants of appropriate pairs of substrates. It was found that the substrate reactivity, as measured by k23/KEA, increase with the number and strength of the secondary interactions in a manner consistent with the binding scheme which has been proposed on the basis of crystallographic studies. The effect of a particular interaction on k23 and on KEA is dependent on the nature of the other interactions. However, the effect of k23/KEA appears to be independent of the presence of the other interactions and therefore characteristic of that particular interaction. The results for these substrates are compared with those found previously for a series of peptide substrates of the structure Ac-Lxn-... Lx2-...-Lx1-Gly-NH2 which have the same acyl moiety as the peptide esters studied in this work.     

Lysine-73 is involved in the acylation and deacylation of beta-lactamase

Lysine 73 is a conserved active-site residue in the class A beta-lactamases, as well as other members of the serine penicillin-sensitive enzyme family; its role in catalysis remains controversial and uncertain. Mutation of Lys73 to alanine in the beta-lactamase from Bacillus licheniformis resulted in a substantial reduction in both turnover rate (k(cat)) and catalytic efficiency (k(cat)/K(m)), and a very significant shift in pK(1) to higher pH in the bell-shaped pH-rate profiles (k(cat)/K(m)) for several penicillin and cephalosporin substrates. The increase in pK(1) is consistent with the removal of the positive ammonium group of the lysine from the proximity of Glu166, to which the acid limb has been ascribed. The alkaline limb of the k(cat)/K(m) vs profiles is not shifted appreciably, as might have been expected if this limb reflected the ionization of Lys73 in the wild-type enzyme. The k(cat)/K(m) at the pH optimum for the mutant was down about 200-fold for penicillins and around 10(4) for cephalosporins, compared to the wild-type, suggesting significant differences in the mechanisms for catalysis of penicillins compared to cephalosporins. Burst kinetics were observed with several substrates assayed with K73A beta-lactamase, indicating an underlying branched-pathway kinetic scheme, and rate-limiting deacylation. FTIR analysis was used to determine whether acylation or deacylation was rate-limiting. In general, acylation was the rate-limiting step for cephalosporin substrates, whereas deacylation was rate-limiting for penicillin substrates. The results indicate that Lys73 plays an important role in both the acylation and deacylation steps of the catalytic mechanism. The effects of this mutation (K73A) indicate that Lys73 does not function as a general base in the catalytic mechanism of beta-lactamase. The existence of bell-shaped pH-rate profiles for the K73A variant suggests that Lys73 is not directly responsible for either limb in such plots. It is likely that both Glu166 and Lys73 are important to each other in terms of maintaining the optimum electrostatic environment for fully efficient catalytic activity to occur.     

Comparison of the kinetic specificity of subtilisin and thiolsubtilisin toward n-alkyl p-nitrophenyl esters.

The p-nitrophenyl esters of straight-chain fatty acids were used as substrates of the enzyme subtilisin Novo (EC 3.4.4.16) and its chemically produced artificial enzyme thiolsubtilisin. Subtilisin and thiolsubtilisin pH--activity profiles were determined, and kinetic effects of the active site O-S substitution were observed. Among the substrates tested, both enzymes show highest specificity with p-nitrophenyl butyrate. It was also found that subtilisin is more sensitive to changes in substrate chain length than is thiolsubtilisin. Second-order acylation rate constants (k2/Ks) are remarkably similar for both enzymes. However, thiolsubtilisin deacylation rate constants and Km values are lower than analogous subtilisin constants. While thiolsubtilisin deacylation rate constants give a pH profile identical with that of subtilisin, the pH profile of thiolsubtilisin acylation rate constants shows an active site pK value lowered from the subtilisin pK of 7.15 and exhibits an inflection point at pH 8.45, which is absent in subtilisin.     

Alcoholysis catalyzed by Candida antarctica lipase B in a gas/solid system: effects of water on kinetic parameters

The influence of water on the kinetics of alcoholysis of methyl propionate and n-propanol catalyzed by immobilized lipase B from Candida antarctica was studied in a continuous solid/gas reactor. In this reactor, the solid phase is composed of a packed enzymatic sample which is percolated by gaseous nitrogen, simultaneously carrying gaseous substrates to the enzyme while removing reaction products. In this system, interactions between the enzyme and nonreacting molecules are avoided, since no solvent is present, and it is thus more easy to assess the role of water. To this end, alcohol inhibition constant, substrates dissociation constants as well as acylation rate constant and ratio of acylation to deacylation rate constants have been determined as a function of water activity (a(w)). Data obtained highlight that n-propanol inhibition constant and dissociation constant of methyl propionate are a lot affected by a(w) variations whereas water has no significant effect on the catalytic acylation step nor on the ratio of acylation to deacylation rate constants. These results suggest the water-independent character of the transition step.     

Activity and conformational changes of alpha-chymotrypsin in reverse micelles studied by spin labeling.

alpha-Chymotrypsin (CT), spin-labeled at the active site by using an acylating label which constitutes a substrate for this protein, has been investigated in reverse micelles formed by AOT in isooctane. The electron spin resonance spectra provided information on conformation, dynamics and deacylation activity. The dynamics of the label bound to CT appears to be more hindered in reverse micelles than in aqueous solution, probably owing to the effect of the micellar environment on protein conformation. The deacylation rate in reverse micelles does not show the characteristic bell-shaped dependence on water content which is generally found for CT enzymatic activity.     

Reaction of tissue-type plasminogen activator with 4-methylumbelliferyl-p-guanidinobenzoate hydrochloride

It has recently been reported that the fluorogenic serine proteolytic active site titrant, 4-methyl-umbelliferyl-p-guanidinobenzoate (MUGB), cannot be employed in this capacity for tissue-type plasminogen activator (TPA) [Geiger, M., and Binder, B.R. (1987) Biochim. Biophys. Acta 912, 34-40]. Since this observation has such important ramifications in this area of research, we have studied the reaction of MUGB with recombinant (rec)TPA under a variety of experimental conditions and find that MUGB is indeed an effective titrant of rec-two chain TPA (recTCTPA) at 4 degrees, a condition under which the deacylation rate constant is diminished to the point that acylation can be readily observed. The KS for the interaction of MUGB with recTCTPA is 43 microM-46 microM, the acylation rate constant, k2, is approximately 3.6 min-1-4.2 min-1, and the rate constant for deacylation of p-guanidinobenzoyl-recTCTPA is 0.084 min-1-0.110 min-1. This same recTCTPA, after treatment with diisopropylfluorophosphate, does not react with MUGB. Single-chain TPA (recSCTPA) has been found to acylate more slowly than its two-chain counterpart and to exhibit a higher degree of turnover of the acyl-enzyme with this reagent. These results demonstrate that the active site concentration of TCTPA can be accurately determined by titration with MUGB, a consideration which is essential to the proper kinetic evaluation of this agent and its genetic variants. On the other hand, the presteady state kinetic characteristics for MUGB toward SCTPA are not favorable for its use as a titrant with this form of the enzyme.     

Beta-lactamases as fully efficient enzymes. Determination of all the rate constants in the acyl-enzyme mechanism.

The rate constants for both acylation and deacylation of beta-lactamase PC1 from Staphylococcus aureus and the RTEM beta-lactamase from Escherichia coli were determined by the acid-quench method [Martin & Waley (1988) Biochem. J. 254, 923-925] with several good substrates, and, for a wider range of substrates, of beta-lactamase I from Bacillus cereus. The values of the acylation and deacylation rate constants for benzylpenicillin were approximately the same (i.e. differing by no more than 2-fold) for each enzyme. The variation of kcat./Km for benzylpenicillin with the viscosity of the medium was used to obtain values for all four rate constants in the acyl-enzyme mechanism for all three enzymes. The reaction is partly diffusion-controlled, and the rate constant for the dissociation of the enzyme-substrate complex has approximately the same value as the rate constants for acylation and deacylation. Thus all three first-order rate constants have comparable values. Here there is no single rate-determining step for beta-lactamase action. This is taken to be a sign of a fully efficient enzyme.     

Cholesterol esterase catalyzed hydrolysis of mixed micellar thiophosphatidylcholines: a possible charge-relay mechanism

Mechanistic features of cholesterol esterase catalyzed hydrolysis of two thiophospholipids, rac-1-(hexanoylthio)-2-hexanoyl-3-glycerophosphorylcholine (6TPC) and rac-1-(decanoylthio)-2-decano-yl-3-glycerophosphorylcholine (10TPC), have been characterized. The hydrolysis of 10TPC that is contained in mixed micelles with Triton X-100 occurs strictly at the micellar interface, since the reaction rate is independent of the micelle concentration but depends hyperbolically on the mole fraction of the substrate in the micelles. This latter observation allows one to calculate the interfacial kinetic parameters V*max and K*m. The hydrolyses of 10TPC and p-nitrophenyl butyrate are similarly inhibited by the transition state analogue inhibitor phenyl-n-butylborinic acid, and therefore, physiological and nonphysiological substrates are processed at the same active site. The similarity of k*cat values for the acyl-similar substrates 10TPC and p-nitrophenyl decanoate indicates that the phospholipase A1 activity of cholesterol esterase is partially rate limited by turnover of a decanoyl-enzyme intermediate. Solvent isotope effects on V*max and V*max/K*m (which monitors acylation only) are approximately 2-3 and are consistent with transition states that are stabilized by general acid-base proton transfers. Proton inventories of V*max/K*m indicate that simultaneous proton transfers stabilize the acylation transition state, which requires a multifunctional acid-base machinery (perhaps a charge-relay system) in the cholesterol esterase active site. Similar results are obtained for the 6TPC reaction, both in the presence and absence of Triton X-100 micelles.     

Chemical mechanism of a cysteine protease, cathepsin C, as revealed by integration of both steady-state and pre-steady-state solvent kinetic isotope effects

Cathepsin C, or dipeptidyl peptidase I, is a lysosomal cysteine protease of the papain family that catalyzes the sequential removal of dipeptides from the free N-termini of proteins and peptides. Using the dipeptide substrate Ser-Tyr-AMC, cathepsin C was characterized in both steady-state and pre-steady-state kinetic modes. The pH(D) rate profiles for both log k cat/ K m and log k cat conformed to bell-shaped curves for which an inverse solvent kinetic isotope effect (sKIE) of 0.71 +/- 0.14 for (D)( k cat/ K a) and a normal sKIE of 2.76 +/- 0.03 for (D) k cat were obtained. Pre-steady-state kinetics exhibited a single-exponential burst of AMC formation in which the maximal acylation rate ( k ac = 397 +/- 5 s (-1)) was found to be nearly 30-fold greater than the rate-limiting deacylation rate ( k dac = 13.95 +/- 0.013 s (-1)) and turnover number ( k cat = 13.92 +/- 0.001 s (-1)). Analysis of pre-steady-state burst kinetics in D 2O allowed abstraction of a normal sKIE for the acylation half-reaction that was not observed in steady-state kinetics. Since normal sKIEs were obtained for all measurable acylation steps in the presteady state [ (D) k ac = 1.31 +/- 0.04, and the transient kinetic isotope effect at time zero (tKIE (0)) = 2.3 +/- 0.2], the kinetic step(s) contributing to the inverse sKIE of (D)( k cat/ K a) must occur more rapidly than the experimental time frame of the transient kinetics. Results are consistent with a chemical mechanism in which acylation occurs via a two-step process: the thiolate form of Cys-234, which is enriched in D 2O and gives rise to the inverse value of (D)( k cat/ K a), attacks the substrate to form a tetrahedral intermediate that proceeds to form an acyl-enzyme intermediate during a proton transfer step expressing a normal sKIE. The subsequent deacylation half-reaction is rate-limiting, with proton transfers exhibiting normal sKIEs. Through derivation of 12 equations describing all kinetic parameters and sKIEs for the proposed cathepsin C mechanism, integration of both steady-state and pre-steady-state kinetics with sKIEs allowed the provision of at least one self-consistent set of values for all 13 rate constants in this cysteine protease's chemical mechanism. Simulation of the resulting kinetic profile showed that at steady state approximately 80% of the enzyme exists in an active-site cysteine-acylated form in the mechanistic pathway. The chemical and kinetic details deduced from this work provide a potential roadmap to help steer drug discovery efforts for this and other disease-relevant cysteine proteases.     

Kinetics of beta-lactam interactions with penicillin-susceptible and -resistant penicillin-binding protein 2x proteins from Streptococcus pneumoniae. Involvement of acylation and deacylation in beta-lactam resistance.

Kinetic interactions of beta-lactam antibiotics such as penicillin-G and cefotaxime with normal, penicillin-susceptible PBP2x from Streptococcus pneumoniae and a penicillin-resistant PBP2x (PBP2x(R)) from a resistant clinical isolate (CS109) of the bacterium have been extensively characterized using electrospray mass spectrometry coupled with a fast reaction (quench flow) technique. Kinetic evidence for a two-step acylation of PBP2x by penicillin-G has been demonstrated, and the dissociation constant, K(d) of 0.9 mm, and the acylation rate constant, k(2) of 180 s(-1), have been determined for the first time. The millimolar range K(d) implies that the beta-lactam fits to the active site pocket of the penicillin-sensitive PBP rather poorly, whereas the extremely fast k(2) value indicates that this step contributes most of the binding affinity of the beta-lactam. The values of K(d) (4 mm) and k(2) (0.56 s(-1)) were also determined for PBP2x(R). The combined value of k(2)/K(d), known as overall binding efficiency, for PBP2x(R) (137 m(-1) s(-1)) was over 1000-fold slower than that for PBP2x (200,000 m(-1) s(-1)), indicating that a major part is played by the acylation steps in penicillin resistance. Most of the decreased binding efficiency of PBP2x(R) comes from the decreased ( approximately 300-fold) k(2). Kinetic studies of cefotaxime acylation of the two PBP2x proteins confirmed all of the above findings. Deacylation rate constants (k(3)) for the third step of the interactions were determined to be 8 x 10(-6) s(-1) for penicilloyl-PBP2x and 5.7 x 10(-4) s(-1) for penicilloyl-PBP2x(R), corresponding to over 70-fold increase of the deacylation rate for the resistant PBP2x(R). Similarly, over 80-fold enhancement of the deacylation rate was found for cefotaxime-PBP2x(R) complex (k(3) = 3 x 10(-4) s(-1)) as compared with that of cefotaxime-PBP2x complex (3.5 x 10(-6) s(-1)). This is the first time that such a significant increase of k(3) values was found for a beta-lactam-resistant penicillin-binding protein. These data indicate that the deacylation step also plays a role, which is much more important than previously thought, in PBP2x(R) resistance to beta-lactams.     

Rate-determining step of butyrylcholinesterase-catalyzed hydrolysis of benzoylcholine and benzoylthiocholine. Volumetric study of wild-type and D70G mutant behavior.

The rate-limiting step for hydrolysis of the positively charged oxoester benzoylcholine (BzCh) by human butyrylcholinesterase (BuChE) is deacylation (k(3)), whereas it is acylation (k(2)) for hydrolysis of the homologous thioester benzoylthiocholine (BzSCh). Steady-state hydrolysis of BzCh and BzSCh by wild-type BuChE and its peripheral anionic site mutant D70G was investigated at different hydrostatic pressures, which allowed determination of volume changes associated with substrate binding, and the activation volumes for the chemical steps. A differential nonlinear pressure-dependence of the catalytic parameters for hydrolysis of both substrates by both enzymes was shown. Nonlinearity of the plots may be explained in terms of compressibility changes or rate-limiting changes. To distinguish between these two possibilities, enzyme phosphorylation by diisopropylfluorophosphate (DFP) in the presence of substrate (BzSCh) under pressure was studied. There was no pressure dependence of volume changes for DFP binding or for phosphorylation of either wild-type or D70G. Analysis of the pressure dependence for steady-state hydrolysis of substrates, and for phosphorylation by DFP provided evidence that no enzyme compressibility changes occurred during the catalyzed reactions. Thus, the nonlinear pressure dependence of substrate hydrolysis reflects changes in the rate-limiting step with pressure. Change in rate-determining step occurred at a pressure of 100 MPa for hydrolysis of BzCh by wild-type and at 75 MPa for D70G. For hydrolysis of BzSCh the change occurred at higher pressures because k(2) < k(3) at atmospheric pressure for this substrate. Elementary volume change contributions upon initial binding, productive binding, acylation and deacylation were calculated from the pressure differentiation of kinetic constants. This analysis shed light on the molecular events taking place along the hydrolysis pathways of BzCh and BzSCh by wild-type BuChE and the D70G mutant. In addition, volume change differences between wild-type and D70G provided new evidence that residue D70 in the peripheral site controls hydration of the active site gorge and the dynamics of the water molecule network during catalysis. Finally, a steady-state kinetic study of the oxyanion hole mutant (G117H) showed that substitution of the ethereal sulfur for oxygen in the substrate alters the final adjustment of substrate in the active site and stabilization of the acylation transition state.     

The effect of monovalent cations on the pre-steady state reaction kinetics of bovine activated plasma protein C and des-1-41-light chain activated plasma protein C

A pre-steady state kinetic analysis of the stimulation by monovalent cations of the activity of bovine activated protein C (APC) and a proteolytic fragment of APC, des-1-41-light chain activated protein C (GDAPC), toward the substrate, 4-methylumbelliferyl p-guanidinobenzoate, has been undertaken. With the cations Na+ and Cs+, at least two cation sites, or classes of sites, on APC were found to be important to the kinetic effects observed. For GDAPC, with both monovalent cations investigated, a single cation-binding site, or class of sites, of kinetic importance was discovered. The most general mechanism that fits all kinetic data was a rapid equilibrium type, with the cation(s) (A) and substrate (S) binding to the enzyme in a random fashion. Cations were found to be essential activators, and only formation of the EAS or EA2S complex led to product generation. For each enzyme, stimulation of the reaction rates was found to be chiefly due to a dramatic enhancement by monovalent cations of the rate constant (k2) for acylation of the enzyme since the dissociation constant (Ks) for enzyme-substrate interactions was increased in the presence of cations, and the deacylation rate constant (k3) was not affected by these activators.     

Methylmalonate-semialdehyde dehydrogenase from Bacillus subtilis: substrate specificity and coenzyme A binding

Methylmalonate-semialdehyde dehydrogenase (MSDH) belongs to the CoA-dependent aldehyde dehydrogenase subfamily. It catalyzes the NAD-dependent oxidation of methylmalonate semialdehyde (MMSA) to propionyl-CoA via the acylation and deacylation steps. MSDH is the only member of the aldehyde dehydrogenase superfamily that catalyzes a β-decarboxylation process in the deacylation step. Recently, we demonstrated that the β-decarboxylation is rate-limiting and occurs before CoA attack on the thiopropionyl enzyme intermediate. Thus, this prevented determination of the transthioesterification kinetic parameters. Here, we have addressed two key aspects of the mechanism as follows: 1) the molecular basis for recognition of the carboxylate of MMSA; and 2) how CoA binding modulates its reactivity. We substituted two invariant arginines, Arg-124 and Arg-301, by Leu. The second-order rate constant for the acylation step for both mutants was decreased by at least 50-fold, indicating that both arginines are essential for efficient MMSA binding through interactions with the carboxylate group. To gain insight into the transthioesterification, we substituted MMSA with propionaldehyde, as both substrates lead to the same thiopropionyl enzyme intermediate. This allowed us to show the following: 1) the pK(app) of CoA decreases by ～3 units upon binding to MSDH in the deacylation step; and 2) the catalytic efficiency of the transthioesterification is increased by at least 10(4)-fold relative to a chemical model. Moreover, we observed binding of CoA to the acylation complex, supporting a CoA-binding site distinct from that of NAD(H).     

Structure and activity of trypsin in reverse micelles

The kinetic properties of trypsin have been studied in reverse micelles formed by two surfactant systems, namely bis(2-ethylhexyl) sodium sulfosuccinate (AOT) in isooctane, and hexadecyltrimethyl ammonium bromide (CTAB) in chloroform/isooctane (1:1, by vol.). Three substrates have been used, namely N alpha-benzoyl-L-Arg ethyl ester, N alpha-benzoyl-L-Phe-L-Val-L-Arg p-nitroanilide (BzPheValArg-NH-Np) in AOT and N alpha-benzyloxycarbonyl-L-Lys p-nitrophenyl ester (ZLysO-Np) in CTAB. One of the main aims of the work was to compare the behaviour of trypsin in reverse micelles with that of alpha-chymotrypsin, for which an enhancement of kcat had been observed with respect to aqueous solutions. The pH profile is not significantly altered in reverse micelles with respect to water, however the kinetic parameters (kcat and Km) differ widely from one another, and are markedly affected by the micellar conditions, in particular by the water content wo (wo = [H2O]/[AOT]). Whereas in the case of BzPheValArg-NH-Np kcat is much smaller than in water, in the case of ZLysO-Np at pH 3.2 (but not at pH 6.0) a slight enhancement with respect to water is observed. On the basis of rapid kinetic spectrophotometry (stopped-flow) and solvent isotope effect studies, this enhancement is ascribed to a change in the rate-limiting step (acylation rather than hydrolysis). As in the case of alpha-chymotrypsin, the maximal activity is found for all substrates at rather small wo values (below 12), which is taken to suggest that the enzyme works better when is surrounded by only a few layers of tightly bound water. Spectroscopic studies [ultraviolet absorption, circular dichroism (CD) and fluorescence] have been carried out as a function of wo. Whereas the absorption properties are practically unchanged, the CD spectrum in AOT micelles has a lower intensity than in water, which is interpreted as a partial unfolding. The intensity is partly restored when Ca2+ ions are added, indicating that the micellar environment may cause a partial denaturation by depleting it of calcium ions. Fluorescence data show that the emission properties of the protein in reverse micelles match those in aqueous solution at around wo = 13 approx., whereas lambda max shifts towards the red by increasing wo, indicating an exposure of the tryptophan residues and probably an unfolding of the whole protein, at wo values above 15. Finally the reaction between trypsin and its specific macromolecular Kunitz inhibitor from soybeans is studied.(ABSTRACT TRUNCATED AT 400 WORDS)     

Kinetic analysis of the dual phospholipid model for phospholipase A2 action

A kinetic scheme is proposed for the action of cobra venom phospholipase A2 on mixed micelles of phospholipid and the nonionic detergent Triton X-100, based on the "dual phospholipid model." (formula; see text) The water-soluble enzyme binds initially to a phospholipid molecule in the micelle interface. This is followed by binding to additional phospholipid in the interface and then catalytic hydrolysis. A kinetic equation was derived for this process and tested under three experimental conditions: (i) the mole fraction of substrate held constant and the bulk substrate concentration varied; (ii) the bulk substrate concentration held constant and the Triton X-100 concentration varied (surface concentration of substrate varied); and (iii) the Triton X-100 concentration held constant and the bulk substrate concentration varied. The substrates used were chiral dithiol ester analogs of phosphatidylcholine (thio-PC) and phosphatidylethanolamine (thio-PE), and the reactions were followed by reaction of the liberated thiol with a colorimetric thiol reagent. The initial binding (Ks = k1/k-1) was apparently similar for thio-PC and thio-PE (between 0.1 and 0.2 mM) as were the apparent Michaelis constants (Km = (k-2 + k3)/k2) (about 0.1 mol fraction). The Vmax values for thio-PC and thio-PE were 440 and 89 mumol min-1 mg-1, respectively. The preference of cobra venom phospholipase A2 for PC over PE in Triton X-100 mixed micelles appears to be an effect on k3 (catalytic rate) rather than an effect on the apparent binding of phospholipid in either step of the reaction.     

The catalytic mode of cysteine proteinases of papain (C1) family

The Proton Inventory (PI) method has been applied in the hydrolysis of synthetic substrates by papain, chymopapain and stem bromelain, comparing also their corresponding pH-(k(cat)/K(m)) profiles, and it was found: (a) k(cat)/K(m)=k(1), and thus K(S)=k(2)/k(1) is a dynamic equilibrium constant, (b) bowed-downward PI for k(cat)/K(m) exhibiting large inverse SIE, and (c) linear PI exhibiting large normal SIE for K(S), k(2) and k(3). A novel finding of this work is that the association of substrates onto all three studied cysteine proteinases proceeds via a stepwise pathway, in contrast to purely concerted pathways found previously for both acylation and deacylation. A hydrogen bond, which seems more likely to be developed across a pK(a)-value close to 4.00, connecting [see text] (papain/chymopapain or bromelain numbering), constitutes another novelty of this work.     

Kinetic behavior of Desulfovibrio gigas aldehyde oxidoreductase encapsulated in reverse micelles

We report the kinetic behavior of the enzyme aldehyde oxidoreductase (AOR) from the sulfate reducing bacterium Desulfovibrio gigas (Dg) encapsulated in reverse micelles of sodium bis-(2-ethylhexyl) sulfosuccinate in isooctane using benzaldehyde, octaldehyde, and decylaldehyde as substrates. Dg AOR is a 200-kDa homodimeric protein that catalyzes the conversion of aldehydes to carboxylic acids. Ultrasedimentation analysis of Dg AOR-containing micelles showed the presence of 100-kDa molecular weight species, confirming that the Dg AOR subunits can be dissociated. UV-visible spectra of encapsulated Dg AOR are indistinguishable from the enzyme spectrum in solution, suggesting that both protein fold and metal cofactor are kept intact upon encapsulation. The catalytic constant (k(cat)) profile as a function of the micelle size W(0) (W(0)=[H(2)O]/[AOT]) using benzaldehyde as substrate showed two bell-shaped activity peaks at W(0)=20 and 26. Furthermore, enzymatic activity for octaldehyde and decylaldehyde was detected only in reverse micelles. Like for the benzaldehyde kinetics, two peaks with both similar k(cat) values and W(0) positions were obtained. EPR studies using spin-labeled reverse micelles indicated that octaldehyde and benzaldehyde are intercalated in the micelle membrane. This suggests that, though Dg AOR is found in the cytoplasm of bacterial cells, the enzyme may catalyze the reaction of substrates incorporated into a cell membrane.     

A rapid-kinetic study of the class C beta-lactamase of Enterobacter cloacae 908R.

The individual rate constants for acylation and deacylation (k2 and k3, respectively) of the class C beta-lactamase of Enterobacter cloacae 908R by ampicillin and carbenicillin have been determined. For several other beta-lactams, the value of k2 was too high to be determined and the k2/k3 ratio could be larger than 10,000. Branched pathways were also shown to occur with several penicillins and cephalosporins.     

The kinetics of hydrolysis of some extended N-aminoacyl-L-arginine methyl esters by porcine pancreatic kallikrein. A comparison with human plasma Kallikrein.

The effects of subsite interactions in the S2-S4 region [Schechter & Berger (1967) Biochem. Biophys. Res. Commun. 27, 157-162] of porcine pancreatic kallikrein (EC 3.4.21.8) on its catalytic efficiency have been investigated. Kinetic constants (Kcat, Km) have been determined for a series of seven extended N-aminoacyl-L-arginine methyl esters whose sequence is based on either the C-terminal sequence of kallidin (-Pro-Phe-Arg) or (-Gly-)nArg. With these substrates it has been found that neither acylation nor deacylation of the enzyme is rate-limiting. Values of Kcat. range from 21.5 to 2320s-1, indicating that there are interactions with different residues in the N-aminoacyl chain and enzyme subsites in the S2-S4 region. It is shown that possible hydrogen-bonded interactions with the enzyme in the S3-S4 region have a significant effect on catalysis. The presence of L-phenylalanine at P2 has a very large effect on both Kcat, and Km, giving a greatly enhanced catalytic efficiency. Substrates with L-proline at P3 also have a marked effect, but in this case the overall effect is one of lowered catalytic efficiency. By comparison with the results of a similar study with human plasma kallikrein I (EC 3.4.21.8), it has been possible to demonstrate that there are considerable differences in kinetic behaviour between the two enzymes. These are related to relative differences in the rates of acylation and deacylation with ester substrates and also the roles of subsites S2 and S3 of the two enzymes.