Biomedical subjects

G L Nelsestuen

Publications and source records attributed to G L Nelsestuen.

At least 19 recordsLinked to original sources

PubMed2001

Reduced outer membrane permeability of Escherichia coli O157:H7: suggested role of modified outer membrane porins and theoretical function in resistance to antimicrobial agents.

Outer membrane permeability of Escherichia coli O157:H7 was determined by an in vivo kinetic model with the periplasmic enzyme alkaline phosphatase [Martinez et al. (1996) Biochemistry 35, 1179-1186]. p-Nitrophenyl phosphate (PNPP) substrate, added to intact bacteria, must diffuse through the outer membrane to reach the enzyme. At low substrate concentration the bacterium was in the perfectly reactive state where all molecules that entered the periplasm were captured and converted to product. Transmembrane diffusion was rate limiting, and the permeability of the outer membrane was determined from kinetic properties. The O157:H7 strain grown at 30 degrees C showed one-sixth the permeability of wild-type E. coli grown at 30 degrees C. Wild-type bacteria grown at >/=37 degrees C show a physiological response with a shift in expression of outer membrane porins that lowered permeability to PNPP by approximately 70%. The O157:H7 strain did not display this temperature-sensitive shift in permeability even though a change in porin expression could be visualized by staining intensity of Omp F and Omp C on acrylamide gels. Altered behavior of the O157:H7 membrane was also indicated by a several thousand-fold lower response to transformation relative to wild-type E. coli. Matrix-assisted laser desorption ionization time of flight mass spectrometry and electrospray ionization mass spectrometry confirmed the expression of the Omp F and Omp C variants that are unique to E. coli O157:H7. This reduced outer membrane permeability can contribute to enhanced resistance of O157:H7 to antimicrobial agents.

Amino Acid Sequence↗

PubMed2001

Elevated function of blood clotting factor VIIa mutants that have enhanced affinity for membranes. Behavior in a diffusion-limited reaction.

Blood clotting factor VIIa is involved in the first step of the blood coagulation cascade, as a membrane-associated enzyme in complex with tissue factor (TF). Factor VIIa is also an important therapeutic agent for hemophilia where its function may include TF-independent as well as TF-dependent mechanisms. This study compared the activity of wild type factor VIIa (WT-VIIa) with that of a mutant with elevated affinity for membrane (P10Q/Q32E, QE-VIIa). Phospholipid and cell-based assays showed the mutant to have up to 40-fold higher function than WT-VIIa in both TF-dependent and TF-independent reactions. Tissue factor-dependent reactions displayed the maximum enhancement when binding had reached equilibrium in competition with another TF-binding protein. In liposome-based assays, the association rate of WT-VIIa with TF occurred at a physical maximum and could not be improved by site-directed mutagenesis. A practical consequence was identical function of WT-VIIa and QE-VIIa in assays that depended entirely on assembly kinetics. Thus, factor VIIa mutants provided unique reagents for probing the mechanism of factor VIIa action. They may also offer superior agents for therapy.

Animals↗

PubMed2000

Vitamin K-dependent proteins.

Vitamin K is required for the synthesis of gamma-carboxyglutamate (Gla) during postribosomal protein modification. Substrates include blood clotting proteins, bone proteins, cell signaling, and receptor proteins. In addition, Gla is a component of short toxin peptides from the marine snail Conus. Studies of structure-function relationships are the most advanced for the blood coagulation proteins. Reviews of vitamin K action and blood coagulation are presented. Special focus is on the structure-function role of Gla in blood coagulation and the impact of this amino acid on enzyme reaction kinetics. This amino acid forms calcium and membrane binding sites for these proteins. Two proposed mechanisms of protein-membrane attachment are reviewed. One involves membrane attachment by protein insertion into the hydrocarbon region of the membrane, while another considers attachment by specific interactions with phospholipid head groups. Membrane attachment generates the potential for several forms of nonclassical enzyme kinetic behaviors, all of which have been observed in vitro. For example, the reaction may be limited by properties of the enzyme active site, a condition that allows use of classic steady-state enzyme kinetic parameters. However, the reaction may be limited by substrate binding to the membrane, by substrate flux through solution, and/or by solvent flow rates across the membrane surface. These states provide special mechanisms that are not anticipated by classical steady-state kinetic derivations. They may be used to regulate coagulation in vivo. Overall, vitamin K research spans the spectrum of biological research and experience. Exciting new ideas and findings continue to emanate from vitamin K-related research.

Animals↗

PubMed1999

Steady-state enzyme kinetics in the Escherichia coli periplasm: a model of a whole cell biocatalyst.

This study provided analysis of in vivo enzyme kinetics in a model system which consisted of alkaline phosphatase in the periplasm of Escherichia coli. Modeling of complete substrate titration curves was achieved for a wide range of intraperiplasmic enzyme levels and outer membrane permeabilities. The results helped to identify the features most important to optimize in vivo reaction velocity. For many situations, a surprising finding was that maximum enzyme expression was not a major concern. For example, for moderate enzyme expression levels and moderate substrate levels (ca 0-5 mM), the limiting step for the enzyme in the periplasm was substrate (para-nitrophenylphosphate) diffusion through the outer membrane. In vivo reaction velocity was directly proportional to substrate concentration, outer membrane permeability, and the cell concentration. Velocity was also quite insensitive to a potent inhibitor of the enzyme. Even though diffusion-limited, periplasmic reaction velocity was quite sensitive to temperature, suggesting that the conformation of porin proteins in the E. coli outer membrane governed the average size of the pore. This model system therefore defined important features of bacterial whole cell biocatalyst design, which may also apply to other reactors using intact cells as catalysts.

Alkaline Phosphatase↗

PubMed1999

Significance of reduced dimensionality in reaction kinetics: impact of multi-site particles.

This review examines novel kinetic properties of enzymes on membrane surfaces or states of restricted diffusion. A leading feature is the presence of multiple enzymes and/or substrates per particle. In these states, enzymes can be influenced by parameters such as the number of substrates or enzymes per particle, particle size, the rates of exchange of substrate or enzyme from the particle, or substrate diffusion to the particle. These steps are independent of the enzyme site parameters which are described by classical enzymology. The results make it clear that non-classical behaviors are important to biological systems, are the basis for some enzyme expression levels and are determinants of cellular design. To identify more unique functions of these states, descriptions of catalysis in the non-solution state should become a part of kinetic education in biology.

Cell Membrane↗

PubMed1999

Membrane association with multiple calcium ions: vitamin-K-dependent proteins, annexins and pentraxins.

Peripheral membrane association with high calcium stoichiometry is shared by three families of proteins: annexins, pentraxins and vitamin-K-dependent proteins. Recent crystal structure determinations, biophysical studies of membrane binding and analyses of protein electrostatic properties offer striking and different concepts for membrane association by each of these protein families.

1-Carboxyglutamic Acid↗

PubMed1999

Enhancement of vitamin-K-dependent protein function by modification of the gamma-carboxyglutamic acid domain: studies of protein C and factor VII.

Vitamin-K-dependent proteins are found in both the pro- and anti-coagulation cascades, and their use in coagulation therapies is expanding rapidly. The vitamin-K-dependent, gamma-carboxyglutamic acid (Gla)-containing regions of proteins in this family are homologous and are responsible for membrane association. Site-directed mutations that enhance the membrane affinity of protein C, an anticoagulant, and of factor VII, a procoagulant, have been identified. These protein C and Factor VII mutants show enhanced activity in many assays, offering opportunities to study the role of membrane in blood clotting reactions and proteins that may have greater therapeutic value.

1-Carboxyglutamic Acid↗

PubMed1998

Enhancement of human protein C function by site-directed mutagenesis of the gamma-carboxyglutamic acid domain.

This study reports properties of site-directed mutants of human protein C that display enhanced calcium and/or membrane binding properties. Mutants containing the S11G modification all showed increased affinity for membranes at saturating calcium concentration. Ser-11 is unique to human protein C, whereas all other vitamin K-dependent proteins contain glycine. This site is located in a compact region of the protein, close to a suggested membrane contact site. Additional changes of H10Q or S12N resulted in proteins with lower calcium requirement for membrane contact but without further increase in membrane affinity at saturating calcium. Mutations Q32E and N33D did not, by themselves, alter membrane affinity to a significant degree. These mutations were included in other mutant proteins and may contribute somewhat to higher function in these mutants. This family of mutants helped discriminate events that are necessary for protein-membrane binding. These include calcium binding to the free protein and subsequent protein-membrane contact. Depending on conditions of the assay used, the mutants displayed increased activity of the corresponding activated protein C (APC) derivatives. The degree of enhanced activity (up to 10-fold) was dependent on the concentration of phospholipid and quality of phospholipid (+/- phosphatidylethanolamine) used in the assay. This was expected, because APC is active in its membrane-associated form, which can be regulated by changes in either the protein or phospholipid. As expected, the largest impact of the mutants occurred at low phospholipid concentration and in the absence of phosphatidylethanolamine. The anticoagulant activity of all proteins was stimulated by protein S, with the greatest impact on the enhanced mutants. Whereas plasma containing Factor V:R506Q was partially resistant to all forms of APC, the enhanced variants were more active than normal APC. Protein C variants with enhanced function present new reagents for study of coagulation and may offer improved materials for biomedical applications.

1-Carboxyglutamic Acid↗

PubMed1998

Manipulation of the membrane binding site of vitamin K-dependent proteins: enhanced biological function of human factor VII.

Recent studies suggested that modification of the membrane contact site of vitamin K-dependent proteins may enhance the membrane affinity and function of members of this protein family. The properties of a factor VII mutant, factor VII-Q10E32, relative to wild-type factor VII (VII, containing P10K32), have been compared. Membrane affinity of VII-Q10E32 was about 20-fold higher than that of wild-type factor VII. The rate of autoactivation VII-Q10E32 with soluble tissue factor was 100-fold faster than wild-type VII and its rate of activation by factor Xa was 30 times greater than that of wild-type factor VII. When combined with soluble tissue factor and phospholipid, activated factor VII-Q10E32 displayed increased activation of factor X. Its coagulant activity was enhanced in all types of plasma and with all sources of tissue factor tested. This difference in activity (maximum 50-fold) was greatest when coagulation conditions were minimal, such as limiting levels of tissue factor and/or phospholipid. Because of its enhanced activity, factor VII-Q10E32 and its derivatives may provide important reagents for research and may be more effective in treatment of bleeding and/or clotting disorders.

Animals↗

PubMed1997

Enhancing the activity of protein C by mutagenesis to improve the membrane-binding site: studies related to proline-10.

Bovine and human protein C show high homology in the amino acids of their GLA domains (amino-terminal 44 residues), despite the about 10-fold higher membrane affinity of the human protein. A proposed membrane contact site and mechanism suggested that this difference was largely due to the presence of proline at position 10 of bovine protein C versus histidine at position 10 of human protein C [McDonald, J.F., Shah, A.M., Schwalbe, R.A., Kisiel, W., Dahlback, B., and Nelsestuen, G.L. (1997) Biochemistry, 36, 5120-5127]. This study examined the impact of replacing proline-10 in bovine protein C with histidine, and the reverse change in human protein C. In both cases, the protein containing proline-10 showed lower membrane affinity, about 10-fold lower for bovine protein C and 5-fold lower for human protein C. As expected, activated human protein C (hAPC) containing proline at position 10 showed 2.4-3.5-fold lower activity than wild type hAPC, depending on the assay used. Most interesting was that bovine APC containing histidine-10 displayed up to 15-fold higher activity than wild type bAPC. This demonstrated the ability to improve both membrane contact and activity by mutation. This general strategy should be applicable to other vitamin K-dependent proteins, providing opportunities to study function as well as to produce proteins that may find use as promoters and inhibitors of blood coagulation in pathological states.

Amino Acid Sequence↗

PubMed1997

Ionic properties of membrane association by vitamin K-dependent proteins: the case for univalency.

Ionic properties of membrane interaction by prothrombin, protein Z, and other vitamin K-dependent proteins were studied to determine the relevance of a monovalent membrane contact mechanism between one phospholipid headgroup and a calcium-lined pore in the protein [McDonald, J. F., Shah, A. M., Schwalbe, R. A., Kisiel, W., Dahlback, B., and Nelsestuen, G. L. (1997) Biochemistry 36, 5120-5127]. For comparison, multivalent ionic interaction was illustrated by peptides of +3 to +5 net charge and by blood clotting factor V. As expected, the peptides were easily dissociated by salt and gave nominal charge-charge interactions (zazb values) of -13 to -17. Factor V showed much higher binding affinity despite nominal zazb values of about 9. Membrane-bound prothrombin and protein Z showed very low sensitivity to salt as long as calcium was at saturating levels (zazb values of approximately -1.3 to -1.4), appropriate for univalent ionic attraction. Prothrombin contains +3 charge groups (Lys-2, Lys-11, Arg-10) that are absent from the GLA domain (residues 1-35) of protein Z, while protein Z contains -4 charge groups (Gla-11, Asp-34, Asp-35) that are absent in prothrombin. Thus, similar zazb relationships indicated little role for these surface charges in direct membrane contact. Calcium-saturated protein Z bound to phosphatidylcholine (PC) in a manner which indicated the addition of one calcium ion, bringing the total calcium stoichiometry in the protein-membrane complex to at least 8. Protein Z bound to phosphatidic acid (PA) in a manner suggesting the need for a fully ionized phosphate headgroup, a property expected by ion pairing in an isolated environment. Electrostatic calculations showed that the proposed protein site for phosphate interaction was electropositive. The cluster of hydrophobic amino acids (Phe-5, Leu-6, and Val-9) on the surface of prothrombin was electronegative, suggesting a role in the electrostatic architecture of the GLA domain. Overall, membrane binding by vitamin K-dependent proteins appeared consistent with the formation of an ion pair in an isolated environment.

Amino Acid Sequence↗

PubMed1997

Steady state enzyme velocities that are independent of [enzyme]: an important behavior in many membrane and particle-bound states.

The popular paradigm for biological education in kinetics involves descriptions that are appropriate for soluble enzymes. Derivations seldom present the assumptions on which the fundamental parameter of these kinetics, the site rate constant, is based. This omission can create difficulty for understanding situations where the assumptions are invalid. Membrane- and particle-bound enzymes systems provide several examples. In fact, biological organisms show macroscopic design and enzyme expression levels which suggest utilization of alternative kinetic mechanisms. The role of substrate affinity and enzyme inhibitors is greatly altered, with correlated impact on biomedical and biotechnological designs. Enzymes may perform functions such as isolation of cell contents from the environment, an action that is usually reserved for membranes. These properties can be mimicked but never perfectly replicated in purified systems. This presentation provides a description of some of these behaviors for membrane- or particle-bound enzymes, using an approach that is closely correlated with the manner in which steady state enzyme kinetics are typically presented.

Binding Sites↗

PubMed1997

Potent inhibition of terminal complement assembly by clusterin: characterization of its impact on C9 polymerization.

The interactions of the heterodimeric apolipoprotein and complement inhibitor, clusterin (CL, 80 kDa), with actively assembling terminal complement proteins were characterized. Clusterin inhibited at three sites and by two modes of action. Clusterin inhibited C9 assembly on C5b-8 and C5b-9 and also bound to C5b-7 to prevent membrane attachment. The impact on C5b-9 assembly was the most potent. C9 assembly was monitored by assembly-induced fluorescence changes of C9 labeled with fluorescein isothiocyanate (FITC-C9). Assembly of monomeric FITC-C9 with C5b-8 or C5b-9(1) produced a substantial decrease in fluorescence intensity due to changes in the environment of the probe. Addition of the next subunit of unlabeled C9 produced a further small change. One equivalent of FITC-C9 bound to C5b-8 at low temperatures, but the fluorescence change and addition of more C9 did not occur until the temperaure was increased. Kinetic analysis of the fluorescence change suggested an irreversible, first-order process with an activation energy of 29 kcal/mol (k = 0.12 s(-1) at 25 degrees C). The kinetic properties differed for C9 addition to C5b-9(1) (0.27 s(-1) at 25 degrees C, 21 kcal/mol), indicating that C9 activation occurred at a different or altered site. Clusterin binding to C5b-8-(FITC-C9)1 caused fluorescence quenching similar to that of unlabeled C9, indicating that it bound to the C9 binding site. Clusterin binding to C5b-8 and C5b-9(1) was reversible with affinities that were 2 and 15 times that of C9 for the C5b-8 and C5b-9(1) complexes, respectively. The results suggested that the presence of <10% of the circulating clusterin in its heterodimeric, active form could reduce the rate of complement cytolysis of nucleated cells by 10-fold, and under some conditions by 100-fold or more. This would provide a high level of protection for certain cells and may allow time for action by other inhibitors of complement.

Clusterin↗

PubMed1997

Comparison of naturally occurring vitamin K-dependent proteins: correlation of amino acid sequences and membrane binding properties suggests a membrane contact site.

Membrane-binding properties of human and bovine forms of vitamin K-dependent proteins Z, S, and C were characterized. Each of these proteins showed unique properties and interspecies differences that correlated with specific amino acid sequence variations in the amino-terminal 45 residues. Protein Z showed 100-fold slower membrane binding and dissociation kinetics relative to other vitamin K-dependent proteins that have been tested. This property seemed to correlate with an extra gamma-carboxyglutamic acid (Gla) residue at position 11 of protein Z. The interspecies difference for protein Z consisted of a higher packing density for the bovine protein on the membrane and a 9-fold slower dissociation rate. Higher affinity correlated with Asp at position 34 of bovine protein Z, where the human protein contains Asn. While both protein S species showed high affinity for the membrane, it was significantly greater for the human protein versus bovine protein S. Again, higher affinity correlated with an Asp (vs Asn) at position 34. Protein C was characterized by binding affinities that were 100-1000-fold lower than the other proteins. Low affinity appeared to be related to loss of Gla-32 (homologous to Gla-33 of protein Z). Interspecies differences of protein C appeared to be related to proline at position 10 (homologous to position 11 of protein Z) of bovine protein C, which produced at least 10-fold lower affinity than the human protein. Comparable substitutions at positions homologous to 11, 33, and 34 of protein Z may also underlie membrane binding behaviors of other vitamin K-dependent proteins. The three-dimensional structure of strontium-prothrombin fragment 1 [Seshadri et al. (1994) Biochemistry 33, 1087] shows that these positions are clustered on the protein surface near strontium-8, another possible candidate for membrane contact. A membrane contact mechanism consisting of an isolated protein-lipid ion pair is proposed. Comparison of naturally occurring vitamin K-dependent proteins has provided possible bases for divergent membrane binding and suggested future approaches to determine biological function.

Amino Acid Sequence↗

PubMed1996

Dynamic features of prothrombin interaction with phospholipid vesicles of different size and composition: implications for protein--membrane contact.

The dynamics of prothrombin interaction with membrane vesicles of different size and composition was investigated to ascertain the impact of membrane surface characteristics and particle size on this interaction. Dissociation rates were highly sensitive to membrane composition and varied from about 20/s for membranes of 10% PS to 0.1/s for membranes of 50% PS. Overall affinity also varied by more than two orders of magnitude. Very small differences between prothrombin binding to SUV versus LUV were found. Association with large unilamellar vesicles (LUV of 115 nm diameter) was about 4-fold slower, when expressed on the basis of binding sites, than association with small unilamellar vesicles (SUV, 30 nm diameter) of the same composition. Both reactions proceeded at less than 25% of the collisional limit so that the differences were largely due to intrinsic binding properties. Vesicles of 325 nm diameter showed even slower association velocities. Dissociation rates from LUV were about 2-fold slower than from SUV. Again, these differences arose primarily from intrinsic binding properties. Dissociation conformed to a single first order reaction over a wide range of protein occupancy on the membrane. At very high packing density, the dissociation rate increased by about 2-fold. At equilibrium, prothrombin preferred binding to SUV over LUV by about 2-fold. This very small difference, despite substantial differences in phospholipid headgroup packing and hydrocarbon exposure, appeared inconsistent with an important role for protein insertion into the hydrocarbon region of the membrane. However, prothrombin-membrane interaction may arise from a series of interaction forces that have compensating features at equilibrium. The small differences in prothrombin binding to SUV versus LUV, together with differences in the number of protein binding sites per vesicle, were important to identify mechanisms of substrate delivery to the active site of the prothrombinase enzyme [Lu, Y., & Nelsestuen, G. L. (1996) Biochemistry 35, 8201-8209].

Binding, Competitive↗

PubMed1996

The prothrombinase reaction: "mechanism switching" between Michaelis-Menten and non-Michaelis-Menten behaviors.

Kinetic properties of prothrombinase were investigated as a function of composition and structure of the membrane component. The kinetic properties were quite diverse, giving linear or nonlinear Eadie-Hofstee plots and substrate concentrations at half-maximum velocity ([S]0.5) that varied from 5 to more than 200 nM. This reaction might be described as a "catalytic system" in order to distinguish it from standard models that have been developed to describe the kinetics of soluble enzymes. The latter do not anticipate a key feature of prothrombinase and probably other membrane-bound enzymes, which is the presence of reaction steps that do not contain an enzyme (E) term. At least four kinetic mechanisms can arise from a logical series of steps that may occur during the prothrombinase reaction. All of these mechanisms appeared to contribute to reaction properties under some conditions. In some cases, one mechanism dominated at low substrate concentration and another at high substrate concentration. This change in the course of a titration was referred to as "mechanism switching". Only membranes of low phosphatidylserine (PS) content displayed Michaelis-Menten behavior. Transfer of substrate from the membrane surface to the enzyme was not important so that the enzyme was involved in capture of substrate directly from solution. As PS content increased, transfer of substrate from the membrane surface to the enzyme occurred. In these cases, multiple mechanisms contributed to the reaction so that K(M) and apparent K(M), properties that describe an enzyme active site, were not appropriate, even when Eadie-Hofstee plots were linear. At high PS content, the enzyme captured every substrate molecule that became bound to the same vesicle. Reaction velocity was governed entirely by protein-membrane binding rather than by enzyme properties. Eadie-Hofstee plots were often nonlinear and/or V(max) was less than kcat[E1]. A small impact from collision-limited kinetics was also detected. Small unilamellar vesicles (SUV, 30 nm diameter) gave higher [S]0.5 values than large unilamellar vesicles (LUV, 100 nm diameter) of the same phospholipid composition. There appeared to be two bases for this behavior. First, LUV may provide a better relationship between the phospholipid surface and the enzyme, giving a better substrate binding site. Second, for membranes containing high PS, the number of substrate binding sites per vesicle contributed to the enhanced function of LUV. These studies showed that mechanism-switching was important to prothrombinase reaction in vitro and suggest that various mechanisms, generated by the nature of the membrane, may be an important regulator for prothrombinase behavior in vivo.

Enzyme Activation↗

PubMed1996

Importance of cis-proline 22 in the membrane-binding conformation of bovine prothrombin.

Upon addition of calcium to the metal-free protein, bovine prothrombin displays a conformational change with behavior of a classic trans- to cis-proline isomerization. The change is accompanied by a decrease of the intrinsic protein fluorescence and is essential to creating the membrane-binding conformation of prothrombin. This study showed that an identical conformational change was displayed by a peptide corresponding to residues 1-45 of prothrombin. This peptide contains a single tryptophan that underwent extensive quenching upon calcium addition. The kinetics were slow (t1/2 = 2.7 min at 24 degrees C) and displayed an activation energy of 24 kcal/mol. These properties overlapped precisely with the behavior of bovine prothrombin fragment 1 (residues 1-156). Consistent with studies on prothrombin and other vitamin K-dependent proteins that have been modified or truncated, the 1-45 peptide required about 10-fold higher calcium to elicit these behaviors than did fragment 1. The conformational change was necessary for membrane binding by the 1-45 peptide. The only proline in this sequence is at position 22. This proline is of the trans configuration in a crystallized form of calcium-bovine prothrombin fragment 1 [Soriano-Garcia, M., et al. (1992) Biochemistry 31, 2554]. Unless the protein conformational change is based on another behavior, this study showed that biochemical properties of the protein are inconsistent with structure solutions. Further studies are needed to reconcile structure/function in membrane association. Proline 22 in bovine prothrombin may constitute a useful biochemical marker for the membrane-binding conformation of a vitamin K-dependent protein.

Amino Acid Sequence↗

PubMed1996

Accurate kinetic modeling of alkaline phosphatase in the Escherichia coli periplasm: implications for enzyme properties and substrate diffusion.

Alkaline phosphatase in the periplasm of Escherichia coli presents many of the complex factors that may influence enzymes in vivo. These include an environment that contains a high enzyme concentration, is densely populated with other macromolecules, and is separated from other compartments by a partial diffusion barrier. A previous study provided a partial description of this situation and developed a model that utilized kinetic behavior to estimate the permeability of the outer membrane [Martinez, M. B., et al., (1992) Biochemistry 31, 11500]. This study extends that description to provide a complete model for the enzyme at all substrate levels. Some of the parameters needed for complete modeling include the following: outer membrane permeability to the substrate and product, catalytic efficiency of the enzyme, number of enzymes per cell, and effects of the reaction product (an inhibitor) on the enzyme. The theoretical model fit the data quite well over a wide range of values for each of these parameters. The best fit of theory with experimental data required that the rate constant for product escape from the periplasm was 4-fold greater than that for substrate entry. This correlated with the relative sizes of the substrate and product. The excellent fit of theory and results suggested that alkaline phosphatase and its substrate were unaffected by the solution conditions in the periplasm. That is, the catalytic parameters (kcat and KM), determined for the enzyme in dilute solution, appeared to be unchanged by the conditions in the periplasm. The major factor that altered the kinetic behavior was the combined effect of the permeability barrier and the dense population of enzyme molecules in the periplasm. Given the large impact of these parameters on reaction properties, the excellent fit of theory and results was striking. Overall, this study demonstrated that enzyme action in the complex biological environment can be accurately modeled, if all factors that influence enzyme behavior are known.

Alkaline Phosphatase↗