What can we learn from kinetics?
Kinetic measurements: the transient- and the steady-state.
Kinetic measurements: ensemble and single-molecule approaches.
One-step irreversible reaction
One-step reversible reaction
Irreversible binding reaction
Reversible binding reaction
Two-step irreversible reaction
Two-step reversible reaction
Two-step reversible binding reaction: induced fit
Two-step reversible binding reaction: conformational selection
Alternative binding mode
Multi-site ligand binding
Multi-step reversible reactions
SOME UNDERLYING PHYSICAL PRINCIPLES
The significance of Free Energy, D G Thermal energy Reaction kinetics and the transition state Time scales of binding kinetics: diffusion-controlled reactions The Time Scales of Macromolecular Conformational Changes Force dependence of rate constants The basis of enzyme catalysis ENZYME KINETICS Enzyme catalysis with a single substrate Enzyme catalysis with two substrates Identification of the rate-limiting step The use of isotopes Enzyme activators and inhibitors Allosteric and Cooperative reactions BEYOND ENZYME KINETICS Protein folding Polymerization reactions Nucleic acid dynamics Motor proteins Membrane Transporters, pumps and channels Biological Clocks Kinetics in vivo EXPERIMENTAL STRATEGIES Sample preparation Preliminary characterization Characterization of equilibrium binding constants Steady-state enzyme kinetics Transient kinetic assays Tuning the system for measurement The use of indicators Single-molecule assays INSTRUMENTATION The basic components Mixing techniques Perturbation techniques Flash photolysis Components for optical detection Detection methods DATA ANALYSIS Basic statistical concepts Analysis of linear functions Least-squares fitting to analytical expressions Global fitting to analytical equations Fitting data directly to kinetic models Multi-wavelength analysis SINGLE-MOLECULE KINETICS Introduction Instrumentation Single-molecule force measurements Analysis of single-molecule kinetic data Analysis of complex time trajectories Autocorrelation analysis Single-molecule enzymology KINETIC THINKING: BACK TO THE FUTURE Biochemical time scales DNA binding proteins Induced Fit versus Conformational Selection revisited Appendices Some useful physical constants Kinetic instrument suppliers Kinetic software Use of spreadsheets in kinetic analysis
Clive Bagshaw obtained a BSc in Biochemistry at the University of Birmingham and a PhD from the University of Bristol. His thesis involved investigation of muscle myosin ATPase activity using transient kinetic methods. The resultant kinetic mechanism, proposed together with his supervisor David Trentham, became textbook information. Following post-doctoral periods at the Universities of Pennsylvania and Oxford, he obtained a lectureship at the University of Leicester, where he taught courses on protein structure and function for 30 years. He continued his research of myosin ATPase activity, initially using a home-built stopped-flow apparatus based on the Gutfreund design. Subsequently, his instrument arsenal was extended, in collaboration with other faculty members, to include commercial stopped-flow, quenched-flow, temperature-jump and flash photolysis instruments. His own research was extended into the mechanisms of myosin ATPase regulation by Ca2+ ions and the kinetics of non-muscle myosins. He wrote a short textbook on Muscle Contraction in 1982, which was revised and extended in 1993 to include in vitro motility assays. The latter was inspired by a sabbatical period in Jim Spudich's laboratory at Stanford University. On return to Leicester, he constructed a total internal reflectance fluorescence microscope to study ATP turnover by myosin at the single-molecule level. In the search for suitable fluorescence probes, he also studied the photophysics of a number of variants of green fluorescent proteins. More recently, he has been involved in collaborative research on other cytoskeletal proteins, S100 proteins, splicing factors and DNA-based photonic wires. From year 2000, Clive has been an instructor on the biannual EMBO Practical Course on Transient Kinetics held at the University of Kent. In 2002 he obtained a personal chair in physical biochemistry and retired from Leicester University in 2011. This transition was catalyzed by a sabbatical at the University of California at Santa Cruz in the laboratory of Michael Stone, which led to an honorary research position that provided the academic resources and freedom to write this book on kinetics. He continues to assist in research using single-molecule methods to study telomerases and enjoy the spectacular natural history of the Monterey Bay.
"a unique and innovative contribution to the field... the first
to my knowledge that covers the entire spectrum of approaches, from
the traditional steady state methods to a thorough account of
transient kinetics and rapid reaction techniques, and then on to
the new single molecule techniques... a significant
-Prof. Stephen Halford, University of Bristol
" Enzyme kinetics is regaining its rightful place at the center
of molecular and cellular biology.... Thus, a new generation of
students will benefit from Clive Bagshaw's Biomolecular Kinetics: A
-Jonathon Howard, Eugene Higgins Professor of Molecular Biophysics & Biochemistry, Yale
"Comprehensive and well written."
-Enrico Di Cera, MD, Alice A. Doisy Professor and Chairman, Department of Biochemistry and Molecular Biology, Saint Louis University
"This book is destined to become a standard reference on the
bookshelf of any laboratory with an interest in kinetic approaches
to biology. Not just a 'how to' handbook but also an accessible
primer in the essentials of kinetic theory & practice."
-Prof. Michael Geeves, University of Kent
"This book is a much needed modern update for the underserved
field of biomolecular kinetics. It covers the practical 'how to'
and troubleshooting information for experiments together with the
fundamentals in one place. It is written by a genuine expert in
kinetic techniques, and is beautifully illustrated. It is highly
accessible to readers at all levels, and I highly recommend this
text to anyone wishing to learn more about the power and potential
applications of kinetics techniques."
-Prof. Emma Raven, University of Leicester
"a gem of a textbook which manages to produce a genuinely fresh,
concise yet comprehensive guide through the nuances of biomolecular
kinetics, penned by one of the giants of this complex but
fundamental field of the life sciences"
-Prof. Mark Leake, University of York
"This well written, concise but very comprehensive text will be
the go-to book for those wanting to take advantage of new (and
classic) approaches, and the challenges and the opportunities they
-Bob Callender, Professor of Biochemistry, Albert Einstein College of Medicine
"This is a comprehensive and detailed yet clear and much needed
book. Kinetic methods are applied throughout biological sciences
and for everyone who is using them this is a must-read."
-Per Jemth, Department of Medical Biochemistry and Microbiology, Uppsala University
"Finally, a book that supports a practical entry into kinetics
for enzymologists and other biochemists. The field sorely needs a
solid, accessible kinetics text like this...."
-Bruce A. Palfey, Department of Biological Chemistry, University of Michigan Medical School
"a lively book with a splendid balance between the description
of most recent experimental work and easily comprehensible
theoretical interpretations.... [It] should help to encourage
students to enter this exciting field and help research workers to
expand the range of their endeavours."
-Herbert Gutfreund, University of Bristol