The activation of human mitogen-activated protein kinase kinase 1 (MKK1) is achieved by phosphorylation at Ser218 and Ser222 within a regulatory loop. Partial activation was achieved by replacing these residues with aspartic/glutamic acid. Higher activity was obtained by introducing four acidic residue substitutions in the regulatory loop, indicating that acidic residues in the loop stabilize an active configuration by the introduction of negative charge. Activation of MKK1 is also achieved by deleting residues 44-51, N-terminal to the consensus catalytic core. Although substitution of residues within this segment by alanine does not affect activity, introduction of proline residues elevates kinase activity, indicating that activation results from perturbation of secondary structure within residues 44-51. Pseudosubstrate inhibition, a commonly observed mechanism of kinase regulation, is not operative in this process. Both the acidic substitutions and the N-terminal deletion increase Vmax, V/K(m),ERK2, and V/K(m),ATP, as is also observed following phosphorylation of wild-type MKK1. A synergistic enhancement of these steady-state rate parameters occurs upon combining the mutations, suggesting that conformational changes induced by mutagenesis together mimic those seen upon phosphorylation.

Structural differences between two genetic variants of bovine beta-lactoglobulins (type A and B) in aqueous solutions were characterized using Fourier transform infrared and circular dichroism spectroscopies. To probe differences in structural dynamics, the effects hydrogen-deuterium exchange were also compared for the two proteins. The infrared spectra recorded in H2O solution for the two proteins were nearly identical in the conformationlly sensitive amide I region. The only exceptions were small differences at the band ascribed to a high-wavenumber beta-sheet component near 1693 cm-1 and the band assigned to turns at 1684 cm-1. In contrast, when the proteins were prepared in D2O solution, marked spectral differences were observed at all regions ascribed to beta-sheet and turn structures. These differences are consistent with the structural differences of the two variants at amino acid residues 64 and 118, which are located at a turn and a beta-sheet structure, respectively, as revealed by X-ray crystallographic studies [Monaco et al. (1987) J. Mol. Biol. 197, 695-706]. The circular dichroism spectra for the two proteins were essentially identical, both before and after hydrogen-deuterium exchange. Therefore, hydrogen-deuterium exchange did not alter the proteins' secondary structure. The enhancement of the amide I spectral difference upon hydrogen-deuterium exchange was ascribed to the differences in the structural mobility of the two proteins. Since the rate of exchange was greater for variant A, it was concluded that this variant has greater structural mobility than variant B. These findings indicate that the combination of infrared spectroscopy and hydrogen-deuterium exchange has great potential in characterization of even subtle structural differences in proteins induced by naturally occurring point mutations and/or site-directed mutagenesis.

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