Interfacial Enzyme Kinetics / Edition 1

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Overview

A vast number of biochemical reactions are catalysed by molecules fixed to the surface of membranes (or other biological structures) with molecules in the surrounding solution. The study of the mechanisms at these "Biointerfaces" are becoming increasingly important for the understanding of biological catalysts, such as enzymes. This project is the first book to deal with the physical and chemical principles of an emerging field of science, for which the authors have set the ground-work.
* The first book to deal with this newly emerging area.

* Concentrates on the chemical and physical foundation of enzyme catalysis

* Key area for the deeper understanding of biocatalytic processes

* Examples for proteins and nucleic acids, two central areas of biochemical and bioorganic research

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Editorial Reviews

From the Publisher
"...outlines the principles, rules, and analytic protocols for the kinetic analysis of the catalytic functions of enzymes at interfaces." (SciTech Book News, Vol. 26, No. 2, June 2002)
From The Critics
For advanced students with a good background in biochemistry and physical chemistry, Berg (Uppsala U) and Jain (U. of Delaware) outline the principles, rules, and analytical protocols for the kinetic analysis of the catalytic functions of enzymes at interfaces. They look at accessibility and exchange, the dilemma for the substrate in solution, ensemble behavior in the scooting mode, and other aspects. Annotation c. Book News, Inc., Portland, OR (booknews.com)
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Product Details

  • ISBN-13: 9780471493044
  • Publisher: Wiley
  • Publication date: 2/22/2002
  • Edition number: 1
  • Pages: 328
  • Product dimensions: 7.64 (w) x 9.92 (h) x 0.91 (d)

Read an Excerpt

Interfacial Enzyme Kinetics


By Otto G. Berg Mahendra Kumar Jain

John Wiley & Sons

ISBN: 0-471-49304-X


Chapter One

Why Interfacial Enzymes?

It is a miracle of the shared knowledge that I have an idea translated into symbols on a page and someone can read those and have the same idea appear in their minds. Yet the writing condition is a strange one. Its unique excellence is at the same time its tragic flaw. Even a positive thing casts a shadow as coherence is imposed on raw phenomenology to make the world amenable. It is necessary to impose a linear order on the multi-dimensional world through discrete concepts with boundaries that we may share but not perceive in the same way. Collective Wisdom

A conceptual need for boundaries and interfaces is intrinsic in all attempts to impose order. The discreteness and identity of a space are provided by boundaries and dividing lines. Such interfaces are also critical and obligatory at all hierarchical levels of organization and function in the biosphere. Membranes and interfaces are the organizing principles to contrast with the aqueous environment. As a unit of living organisms, the very existence of a cell is based on membranes and interfaces that identify it as a morphological entity distinct from the rest of the universe.

The subcellular interfaces

If life is impossible without water, it is inconceivable without membrane-water interfaces. The aqueous milieu in organisms is compartmentalized by membranes.Tissues are made up of cells, and the cytoplasmic space in cells is interrupted by organelles. Virtually all processes that sustain an organism, ranging from the morphology to specialized functions, depend on discrete functional identities provided to the space by membranes and interfaces. These hydrophobic barriers of molecular dimensions are less than 10 nm thick compared to the 1000 to 50 000 nm diameter of most cells. An appreciation of the role of interfaces in the form and function of a cell is probably best gained by examining the compartments of a cell as in Figure 1.1. Here all the dark lines represent the bilayer interfaces. All such membrane-bounded structures enclose an aqueous compartment that is distinct in composition from the surrounding medium. Noteworthy features of the heterogeneity in the cellular milieu include:

(a) An eukaryotic cell typically contains one to several thousand copies each of a variety of organelles. Although in metabolic communication, organelles retain their characteristic morphology, composition (lipids, proteins, and other macromolecules) and specialized functions.

(b) The organizational matrix of membranes (the thick lines in Figure 1.1) is a phospholipid bilayer with a hydrophobic interior sandwiched between two polar interfaces in contact with the aqueous phases.

(c) Virtually all phospholipid molecules in a cell are present in the bilayer of membranes. The interface area is twice the area of the bilayer. With a cross-sectional area of about 100 [Å.sup.2](= 1 [nm.sup.2]) per phospholipid molecule, one mole of phospholipid dispersed as bilayerwill have the interface of 600 000 [m.sup.2] (about a quarter of a square mile). Consider the consequences of the fact that the total phospholipid concentration in tissues like brain, kidney, liver and heart is typically 50 mM. Therefore, 1 [cm.sup.3] of a tissue has 30 [m.sup.2] of the phospholipid interface. In other words, with 0.05 millimole of the phospholipid packed within a 1 cm cube, equivalent to 50 mM total concentration, the average thickness of the interspersed aqueous compartments separating 1 [cm.sup.2] slices of the interfaces would be approximately 300 Å with 50 Å thickness for the phospholipid bilayer. (Note that 1 cm = 0.01 m = [10.sup.7] nm = 108 Å.)

(d) The bilayer interface spans a broad range of polarity within a thickness of less than 1 nm. The dielectric constant is 80 in the aqueous phase compared to less than 2 in the hydrophobic interior.

(e) In terms of weight, number or genome length, about half of the protein in cells is membrane-associated.

(f) Membranes act as a workbench for about half of the cellular protein. Membrane enzymes are operationally (Figure 1.2) either matrix enzymes that access their substrate from the bulk aqueous phase or interfacial enzymes that directly access the membrane-localized substrate. The soluble enzymes in the aqueous phase access their substrate directly from the aqueous phase.

1.1 Structural Diversity of Nonpolar and Amphiphilic Solutes

The primary functional distinction between interfacial versus matrix or soluble enzymes comes from the relative tendency of the substrate to be in the aqueous phase or the interface. Such effects assume added importance in the microscopically heterogeneous cellular environment, where the thickness of the aqueous compartment between the interfaces may rarely exceed more than a few hundred Å. In effect, the cytoplasmic aqueous phase is interspersed by stable membranes with a rather steep polarity gradient that spans from the hydrophobic interior of the bilayer to the water-compatible polar surface. Much of the polarity change occurs within 10 Å interface near the bilayer surface. As developed in this book, mechanisms and biophysical strategies that have evolved to deal with the physical reality of the substrate solubility and accessibility are beginning to be understood. As implicit in Figure 1.2, the partitioning (S to S*) and enzyme binding (E to E*) equilibria determine the kinetic path and influence the turnover rate.

The evolutionary significance of biochemical processes at or across interfaces is best appreciated by examining molecular structures of some of the nonpolar and amphiphilic solutes with nonpolar functional groups with or without polar substituents. For example, galactosyl lipids (Figure 1.3) are the most abundant lipids in the biosphere. With a polar polyhydroxylic moiety at one end, the glycolipids provide the bilayer matrix for virtually all the plant membranes. The membrane of Archaebacteria from volcanic springs contains a unique class of bipolar lipids, possibly the rarest of the membrane lipids. Bipolar lipids contain two polar head groups attached at the two ends of the intervening isoprene chains. Thus the 'monolayer' of a bipolar lipid molecule has two polar interfaces as the structural basis for the stability of a membrane that functions at the near-boiling temperatures of volcanic springs. Triglycerides (Figure 1.3) are the predominant components of dietary fat. They are among the most hydrophobic of the naturally occurring lipids. Virtually devoid of a polar group, acylglycerols do not dissolve or disperse in water. Glycerides with long acyl chains are stored as phase-separated droplets in plant seeds to provide food and fuel during germination. In addition to the survival value during starvation, the fat deposits in adipose tissues of animals also provide insulating layers. Properly distributed body fat has also inspired changing conceptions of beauty and health.

In addition to the food and fiber dependence on plants for the survival of all organisms, there is an even broader symbiosis between the phototrophs and chemotrophs. The sustainability of the biosphere at such a fundamental level is based on the ability of plants to use sunlight to fix carbon dioxide into carbohydrates that are used as metabolic fuel and building blocks by virtually all other organisms. At the heart of this cosmic machine driven by the sunlight are the oriented molecules of chlorophyll and other pigments (Figure 1.4) in the chromophores of the photosystem P680 and P700 complex. Such photosynthesis complexes are localized in a virtually crystalline lattice in the thylakoid membrane of chloroplasts and other plastids. They capture photons and transduce a part of their energy to generate the proton concentration (pH) gradient across the membrane. The pH gradient drives the reduction of NADP to NADPH and also the synthesis of ATP. Both of these high-energy products are used for the incorporation of C[O.sub.2] into ribulose-1,5-bisphosphate. Through such pathways C[O.sub.2] is ultimately 'fixed' in glucose and its polymers such as starch (food) and cellulose (fiber).

In addition to their role in the biosynthesis of food and fiber, the roles of hydrophobic solutes as trace components are also impressive. Structurally diverse minor nonpolar metabolites include pigments, steroid hormones, pheromones, eicosanoids and other signal molecules. Of course, their behavioral, developmental and pathophysiological effects are the end result of the exquisite control they exert on processes ranging from macromolecular syntheses to osmoregulation and ion metabolism. Obviously, metabolic pathways have evolved to deal with a large polarity spectrum of biomolecules, where the water-soluble molecules represent only one extreme.

1.2 To Be or Not to Be in Water

Depending on the energetic balance of the interactions between the polar and nonpolar groups of a solute with each other and with water molecules, solutes in the aqueous phase exhibit wide-ranging solution characteristics. One extreme is the ideal solution with molecularly monodispersed solute in the bulk solvent without any solute-solute interactions. At the other extreme, virtually insoluble nonpolar solutes avoid any interaction with the aqueous phase by forming a separate phase. As developed in Chapter 2, complex phase behavior is observed in dispersions of amphipathic solutes with polar as well as nonpolar hydrophobic functional groups in the same molecule.

Accommodation of a solute between the organized water molecules in the aqueous phase depends on the ability of the functional groups of the solute to disrupt water-water interactions by promoting favorable water-solute interactions (Figure 1.5). Ions and most of the oxygen- and nitrogen-containing functional groups are capable of such favorable interactions with the polarized [H.sup.[delta]+] -[O.sup.[delta]+] -[H.sup.[delta]+] molecule that has a fractional negative charge on the electronegative oxygen and fractional positive charges on both hydrogens. As a consequence of the electronegativity of oxygen and nitrogen, solutes with fractional charges on polarized O- and N-containing functional groups have water-solute interactions that are energetically more favorable than the water-water interactions. For the same reasons, solute-solute and solute-water interactions are also at the heart of the interface phenomena as seen in the organization and exchange properties of the nonpolar and amphipathic solutes in aqueous dispersions. Such interactions are critical for understanding the effect of substrate accessibility on the enzyme-catalyzed turnover in the presence of dispersed phases.

1.2.1 Solubility and partitioning

Nonpolar solutes, with fewer polar groups capable of interacting with water, are less likely to compete in the water-water interactions. As a result, the dominant hydrophobic effect leads to lower solubility in the aqueous phase, higher partitioning in a nonpolar phase and a nonideal solution behavior due to the formation of aggregates. For example, above their solubility limit in the aqueous phase, nonpolar solutes phase separate and precipitate. As the basis for the S to S* equilibrium (Figure 1.2) the molecularly dispersed nonpolar solutes in solution favorably partition into nonpolar phases. The equilibrium concentration of a monodisperse nonpolar solute in the aqueous phase, as well as the concentration of the partitioned solute, does not change with total concentration of a solute above its solubility limit (developed further in Chapters 2 to 4).

The solution behavior and the partitioning characteristics of a nonpolar or amphiphilic substrate in a heterogeneous environment introduce the challenge of defining, establishing and evaluating the consequences of the substrate concentration variable for an enzyme-catalyzed reaction. It appears that several strategies have evolved to deal with the physical reality of low solubility and aggregation behavior. For example, sparingly soluble long chain fatty acids in the extracellular milieu bind to serum albumins that 'ferry' nonpolar solutes from tissue to tissue. Such binding proteins also act as 'buffers' by reducing the aqueous phase concentration of nonpolar solutes. Another strategy is that of acyl-coenzyme A derivatives with higher aqueous phase solubility compared to the free fatty acids. Thus the enzyme affinity well above the solubility limit makes the acyl-CoA suitable soluble substrates for fatty acid metabolism within the cell. Both of these mechanisms also solve potential problems associated with the detergent action of ionized free fatty acids (soaps).

1.2.2 Amphiphiles

The term refers to the ambivalence of the aqueous phase for the accommodation with polar and nonpolar regions of a solute molecule such as an ionized fatty acid (Figure 1.4), glycolipids (Figure 1.3) and phospholipids (Figure 1.6). Amphiphiles have a polar (head) region that is separated from the nonpolar (tail) polymethylene chain, and share a common tendency to form organized phases and dispersions. The molecular diversity of amphiphiles is large because specific interactions are not required for the aggregate formation or the partitioning of the solute driven by the hydrophobic effect. In addition to the acyl and alkyl chains of the hydrophobic tail there may be a steroid nucleus or an aromatic hydrocarbon moiety. The hydrophilic character of the head group may come from virtually any hydrogen-bonding or ionic group that interacts with water. The minimum polarity required of a head group of an amphiphile appears to be between a hydroxyl (-C[H.sub.2]OH) and a carboxylate (-CO[O.sup.-]) group. Amphiphiles with weaker polarity form microcrystals above their solubility limit in water. Organized structures, such as bilayers or micelles, are formed in dispersions of amphiphiles with stronger polar groups (sulfate, sulfonate, phosphate, ammonium, carboxylate) or bulky oxygen-containing uncharged groups (amine oxide, polyoxyethylene, phosphine oxide, sugar residue) capable of extensive hydrogen bonding and dipolar interactions with water. As the basis for the interface phenomena, the organizational constraints for an amphiphile in aggregates depend on the polar and nonpolar groups.

1.2.3 Phospholipids

Glycolipid (Figure 1.3) and phospholipid (Figure 1.6) make up the bilayer matrix of membranes (Figure 1.1) in virtually all organisms. Such amphiphiles have acommon structural motif: cylindrical shape with a strongly polar head group at one end of the strongly hydrophobic diacylglycerol moiety. Phospholipids are the diglycerides with a polar sn-3-glycero phosphodiester substituent.

Continues...


Excerpted from Interfacial Enzyme Kinetics by Otto G. Berg Mahendra Kumar Jain Excerpted by permission.
All rights reserved. No part of this excerpt may be reproduced or reprinted without permission in writing from the publisher.
Excerpts are provided by Dial-A-Book Inc. solely for the personal use of visitors to this web site.

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Table of Contents

Preface
Theory Boxes
List of Symbols
Why Interfacial Enzymes?
Interface Phenomena: Accessibility and Exchange
To Be or Not To Be: Dilemma for the Substrate in Solution
Interfacial Processivity: Ensemble Behavior in the Scooting Mode
Analysis of the Processive Reaction Progress
Detailed Balance Conditions for Interfacial Equilibria
Rapid Substrate Replenishment in the Quasi-Scooting Mode
Interfacial Allostery
Inhibition: Specific or Nonspecific
The Delay to the Steady State in the Reaction Progress
Nonidealities of the Dispersed Phases
Effects of Reduction of Dimensionality

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