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Earlier studies of the magnetic field dependence of the nuclear spin magnetic relaxation rate of solvent protons in solutions of diamagnetic proteins have indicated that this dependence (called relaxation dispersion) is related to the rotational Brownian motion of solute proteins. In essence, the dispersion is such that 1/T1 (the proton spin-lattice relaxation rate) decreases monotonically as the magnetic field is increased from a very low value (approximately 10 Oe); the dispersion has a point of inflection at a value of magnetic field which depends on protein size, shape, concentration, temperature, and solvent composition. The value of the proton Larmor precession frequency nu(c) at the inflection field appears to relate to tau (R), the rotational relaxation time of the protein molecules. We have measured proton relaxation dispersions for solutions of various proteins that span a three-decade range of molecular weights, and for one sample of transfer ribonucleic acid. We have also measured deuteron relaxation dispersions for solutions of three proteins: lysozyme, carbonmonoxyhemoglobin, and Helix pomatia hemocyanin with molecular weight 900 000. A quantitative relationship between both proton and deuteron dispersion data and protein rotational relaxation is confirmed, and the point is made that magnetic dispersion measurements are of very general applicability for measuring the rotational relaxation rate of macromolecules in solution. It has been previously shown that the influence of proton motion on the relaxation behavior of the solvent is not due to exchange of solvent molecules between the bulk solvent and a hydration region of the protein. In the present paper, we suggest that the interaction results from a long range hydrodynamic effect fundamental to the situation of large Brownian particles in an essentially continuum fluid. The general features of the proposed mechanism are indicated, but no theoretical computations are presented.

Mechanisms for the ribonuclease T1 (RNase T1; EC 3.1.27.3) catalyzed transesterification reaction generally include the proposal that Glu58 and His92 provide general base and general acid assistance, respectively [Heinemann, U., & Saenger, W. (1982) Nature (London) 299, 27-31]. This view was recently challenged by the observation that mutants substituted at position 58 retain high residual activity; a revised mechanism was proposed in which His40, and not Glu58, is engaged in catalysis as general base [Nishikawa, S., Morioka, H., Kim, H., Fuchimura, K., Tanaka, T., Uesugi, S., Hakoshima, T., Tomita, K., Ohtsuka, E., & Ikehara, M. (1987) Biochemistry 26, 8620-8624]. To clarify the functional roles of His40, Glu58, and His92, we analyzed the consequences of several amino acid substitutions (His40Ala, His40Lys, His40Asp, Glu58Ala, Glu58Gln, and His92Gln) on the kinetics of GpC transesterification. The dominant effect of all mutations is on Kcat, implicating His40, Glu58, and His92 in catalysis rather than in substrate binding. Plots of log (Kcat/Km) vs pH for wild-type, His40Lys, and Glu58Ala RNase T1, together with the NMR-determined pKa values of the histidines of these enzymes, strongly support the view that Glu58-His92 acts as the base-acid couple. The curves also show that His40 is required in its protonated form for optimal activity of wild-type enzyme. We propose that the charged His40 participates in electrostatic stabilization of the transition state; the magnitude of the catalytic defect (a factor of 2000) from the His40 to Ala replacement suggests that electrostatic catalysis contributes considerably to the overall rate acceleration. For Glu58Ala RNase T1, the pH dependence of the catalytic parameters suggests an altered mechanism in which His40 and His92 act as base and acid catalyst, respectively. The ability of His40 to adopt the function of general base must account for the significant activity remaining in Glu58-mutated enzymes.

The magnetic spin-lattice relaxation rates of solvent water nuclei are known to increase upon addition of diamagnetic solute protein. This enhancement of the relaxation rate is a function of magnetic field, and the orientational relaxation time of the protein molecules can be deduced from analysis of the field-dependent relaxation rates. Although the nature of the interactions that convey information about the dynamics of protein motion to the solvent molecules is not established, it is known that there is a contribution to the relaxation rates of solvent protons that plays no role in the relaxation of solvent deuterons and 17O nuclei. We show here that the additional interaction arises from a cross-relaxation process between solvent and solute protons. We introduce a heuristic three-parameter model in which protein protons and solvent protons are considered as two separate thermodynamic systems that interact across the protein-solvent interface. The three parameters are the intrinsic relaxation rates of each system and a cross-relaxation term. The sign of the latter term must always be positive, for all values of magnetic field, in order for magnetization energy to flow from the hotter to the cooler system. We find that the magnetic field-dependence of the cross-relaxation contribution is much like that of the remaining solvent proton relaxation, i.e., about the same as the deuteron relaxation field dependence. This finding is not compatible with the predictions of expressions for the cross-relaxation that have been used by other authors, but not applied to data over a wide range of magnetic field strength. The model predicts that the relaxation behavior of both the protein protons and the solvent protons is the sum of two exponentials, the relative contributions of which would vary with protein concentration and solvent isotopic composition in a fashion suggestive of the presence of two classes of protein protons, when there is in reality only one. This finding has immediate implications for the interpretation of published proton relaxation rates in complex systems such as tissues; these data should be reexamined with cross-relaxation taken into account.