On the function and mechanism of action of peroxidases

The kinetic parameters of enzymes are key to understanding the rate and specificity of most biological processes. Although specific trends are frequently studied for individual enzymes, global trends are rarely addressed. We performed an analysis of kcat and KM values of several thousand enzymes collected from the literature. We found that the “average enzyme” exhibits a kcat of ∼10 s–1 and a kcat/KM of ∼105 s–1 M–1, much below the diffusion limit and the characteristic textbook portrayal of kinetically superior enzymes. Why do most enzymes exhibit moderate catalytic efficiencies? Maximal rates may not evolve in cases where weaker selection pressures are expected. We find, for example, that enzymes operating in secondary metabolism are, on average, ∼30-fold slower than those of central metabolism. We also find indications that the physicochemical properties of substrates affect the kinetic parameters. Specifically, low molecular mass and hydrophobicity appear to limit KM optimization. In accordance, subst...

The Moderately Efficient Enzyme: Evolutionary and Physicochemical Trends Shaping Enzyme Parameters

Surface plasmon resonance biosensors have become increasingly popular for the qualitative and quantitative characterization of the specific binding of a mobile reactant to a binding partner immobilized on the sensor surface. This article reviews the use of this new technique to measure the binding affinities and the kinetic constants of reversible interactions between biological macromolecules. Immobilization techniques, the most commonly employed experimental strategies, and various analytical approaches are summarized. In recent years, several sources of potential artifacts have been identified: immobilization of the binding partner, steric hindrance of binding to adjacent binding sites at the sensor surface, and finite rate of mass transport of the mobile reactant to the sensor surface. Described here is the influence of these artifacts on the measured binding kinetics and equilibria, together with suggested control experiments.

Use of surface plasmon resonance to probe the equilibrium and dynamic aspects of interactions between biological macromolecules.

A theory, which connects rotational brownian motion with intensity fluctuations of the light emitted from fluorescent molecules excited by linearly polarized light, is given. Analysis of rotational diffusion in this way does not depend on the close relationship between fluorescence lifetime and rotational relaxation times, which is necessary in present methods and thus makes an enlarged time range available for fluorescence spectroscopy. When short fluorescence lifetimes are used the rotational diffusion of the molecule in its ground state will be observed.

Rotational brownian motion and fluorescence intensify fluctuations

A method is developed and tested for extracting diffusion‐controlled rate constants for condensed phase bimolecular reactions from Brownian dynamics trajectory simulations. This method will be useful when highly detailed model systems are employed, such as those required to explore the complicated range of interactions between enzymes and their substrates. The method is verified by comparing with exact analytical results for simple cases of spheres with uniform reactivity subject to various centrosymmetric Coulombic and Oseen slip hydrodynamic interactions. The utility of the method is illustrated for more complicated cases involving anisotropic reactivity and rotational diffusion.

Brownian dynamics simulation of diffusion‐influenced bimolecular reactions

Role of orientation constraints and rotational diffusion in bimolecular solution kinetics

Correlation functions have been determined for the fluctuating intensity of the depolarized component of forward-scattered laser light from solutions of DNA. The molecular correlation function of calf thymus DNA (mol wt ∼15 × 106) appears to exhibit a longest relaxation time (τ25,w, ∼ 18 msec) close to what one would predict from the flowdichroism measurements of Callis and Davidson and, in addition, manifests a spectrum of faster times down to tenths of milliseconds. Furthermore, a major fraction of the amplitude of fluctuations in the angular distribution of segment axes is relaxed on a very much shorter time scale (of the order of 20 microseconds) that appears to be relatively insensitive to molecular weight of the DNA, or to near-melting temperatures.



The temperature profile of the longest relaxation time has been obtained and found to exhibit a peculiar spike near Tm, which, together with the absence of a corresponding spike in the (high shear) viscosity, has been interpreted as indicative of an increase in the molecular weight of the DNA in a narrow temperature region near Tm. Correlation functions for polarized light scattered at finite angles were obtained in an attempt to determine the temperature dependence of the translational diffusion coefficient. Although the data contain an extremely slow component that does not admit a simple interpretation, there is some indication of a decrease in the translational diffusion coefficient near Tm, thus supporting the notion of an aggregation occurring near Tm. Finally, a “counterion escape” mechanisn is proposed for the apparent aggregation.

Rotational relaxation of macromolecules determined by dynamic light scattering. II. Temperature dependence for DNA

Orientation constraints and rotational diffusion in bimolecular solution kinetics. A simplification

The intensity autocorrelation function for the depolarized component of forward-scattered light from a solution of large polymeric molecules is derived in terms of the correlation function for the amplitudes of the Y2,±1(θ,ϕ) fluctuations in the anglar distribution of segments in the solution without any assumptions regarding the statistical properties of the scatterad light field. Effects arising from the use of polychromatic incident light and from the mixing of the scattered and polychromatic incident light beams are examined in detail.



Apparatus for observing the depolarized forward-scattered light, digitizing and storing the fluctuating phototube current at rates from 10 to 540,000 times per second, and computing the correlation functions directly in the time-domain is described herein. Correlation functions were obtained for 0.05 mg/ml solution of tobacco mosaic virus at pH 9.1 and also at pH 6. The degree of association of the virus appears to be independent of pH, and the monomer relaxation times (corrected to 25°C) extracted from the data by a least-squares procedure lie in the range 0.44–0.49 msec, also independent of pH. The absence of faster component in the correlation function between 6 μsec and 0.5 msec is used in conjunction with thermal fluctuation theory to infer a lower limit for the effective Young's modulaus of the rod, E ≤ 2.5 × 107 dynes/cm2.

Rotational relaxation of macromolecules determined by dynamic light scattering. I. Tobacco mosaic virus

The dialysis data of Huang and Ts'o for the cooperative binding of adensine to polyuridylic acid are analyzed here using a grand-partition function Ising model method similar to that originally employed for polyelectrolytes by Rice and Nagasawa. An appropriate modification permitting the treatment of the sliding degeneracy of the two polyuridylic acid strands is also included. In addition to the previously estimated stacking energy of about −6 kcal/mole one also obtains the free energy change F for the transfer of a single adenosine molecule from a fixed site in solution to a fixed site on the polyuridylic acid. This binding energy falls in the range F = −140 to +620 cal/mole, indicating that binding in the 1:2 (purine: pyrimidine) complex is either very weak or repulsive. The absence of any comparable cooperative stacking of adenosines in solution at the same concentration together with the likely repulsive character of the binding implies that the stacking energy must contain a significant contribution from other processes than simple stacking of adenosines. A generalization of the theory to treat multicomponent binding and longer-range interactions is effected, and the form applicable to simultaneous binding of both adenosine and guanosine by polyuridylic acid is presented.

Kenneth S. Schmitz

Papers

Role of orientation constraints and rotational diffusion in bimolecular solution kinetics

Rotational relaxation of macromolecules determined by dynamic light scattering. II. Temperature dependence for DNA

Orientation constraints and rotational diffusion in bimolecular solution kinetics. A simplification

Rotational relaxation of macromolecules determined by dynamic light scattering. I. Tobacco mosaic virus

Cooperative binding of adenosine by polyuridylic acid: a further analysis.