The propeptide of cruzipain--a potent selective inhibitor of the trypanosomal enzymes cruzipain and brucipain, and of the human enzyme cathepsin F.

Papain-like cysteine proteases of pathogenic protozoa play important roles in parasite growth, differentiation and host cell invasion. The main cysteine proteases of Trypanosoma cruzi (cruzipain) and of Trypanosoma brucei (brucipain) are validated targets for the development of new chemotherapies. These proteases are synthesized as precursors and activated upon removal of the N-terminal prodomain. Here we report potent and selective inhibition of cruzipain and brucipain by the recombinant full-length prodomain of cruzipain. The propeptide did not inhibit human cathepsins S, K or B or papain at the tested concentrations, and moderately inhibited human cathepsin V. Human cathepsin F was very efficiently inhibited (K(i) of 32 pm), an interesting finding indicating that cruzipain propeptide is able to discriminate cathepsin F from other cathepsin L-like enzymes. Comparative structural modeling and analysis identified the interaction between the beta1p-alpha3p loop of the propeptide and the propeptide-binding loop of mature enzymes as a plausible cause of the observed inhibitory selectivity.

Alterations in the alpha2 isoform of Na,K-ATPase associated with familial hemiplegic migraine type 2.

A number of missense mutations in the Na,K-ATPase alpha2 catalytic subunit have been identified in familial hemiplegic migraine with aura. Two alleles (L764P and W887R) showed loss-of-function, whereas a third (T345A) is fully functional but with altered Na,K-ATPase kinetics. This study describes two additional mutants, R689Q and M731T, originally identified by Vanmolkot et al. [Vanmolkot, K. R., et al. (2003) Ann. Neurol. 54, 360-366], which we show here to also be functional and kinetically altered. Both mutants have reduced catalytic turnover and increased apparent affinity for extracellular K(+). For both R689Q and M731T, sensitivity to vanadate inhibition is decreased, suggesting that the steady-state E(1) <==> E(2) poise of the enzyme is shifted toward E(1). Whereas the K'(ATP) is not affected by the R689Q replacement, the M731T mutant has an increase in apparent affinity for ATP. Analysis of the structural changes effected by T345A, R689Q, and M731T mutations, based on homologous replacements in the known crystal structure of the sarcoplasmic reticulum Ca-ATPase, provides insights into the molecular bases for the kinetic alterations. It is suggested that the disease phenotype is the consequence of lowered molecular activity of the alpha2 pump isoform due to either decreased K(+) affinity (T345A) or catalytic turnover (R689Q and M731T), thus causing a delay in extracellular K(+) clearance and/or altered localized Ca(2+) handling/signaling secondary to reduced activity in colocalized Na(+)/Ca(2+) exchange.

Mirror-image packing in enantiomer discrimination molecular basis for the enantioselectivity of B.cepacia lipase toward 2-methyl-3-phenyl-1-propanol.

Synthetic chemists often exploit the high enantioselectivity of lipases to prepare pure enantiomers of primary alcohols, but the molecular basis for this enantioselectivity is unknown. The crystal structures of two phosphonate transition-state analogs bound to Burkholderia cepacia lipase reveal this molecular basis for a typical primary alcohol: 2-methyl-3-phenyl-1-propanol. The enantiomeric alcohol moieties adopt surprisingly similar orientations, with only subtle differences that make it difficult to predict how to alter enantioselectivity. These structures, along with a survey of previous structures of enzyme bound enantiomers, reveal that binding of enantiomers does not involve an exchange of two substituent positions as most researchers assumed. Instead, the enantiomers adopt mirror-image packing, where three of the four substituents at the stereocenter lie in similar positions. The fourth substituent, hydrogen, points in opposite directions.