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African trypanosomes contain a cyclic derivative of oxidized glutathione, N1,N8-bis(glutathionyl)spermidine, termed trypanothione. This is the substrate for the parasite enzyme trypanothione reductase, a key enzyme in disulfide/dithiol redox balance and a target enzyme for trypanocidal therapy. Trypanothione reductase from these and related trypanosomatid parasites is structurally homologous to host glutathione reductase but the two enzymes show mutually exclusive substrate specificities. To assess the basis of host vs parasite enzyme recognition for their disulfide substrates, the interaction of bound glutathione with active-site residues in human red cell glutathione reductase as defined by prior X-ray analysis was used as the starting point for mutagenesis of three residues in trypanothione reductase from Trypanosoma congolense, a cattle parasite. Mutation of three residues radically alters enzyme specificity and permits acquisition of glutathione reductase activity at levels 10(4) higher than in wild-type trypanothione reductase.

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Lactate dehydrogenase (LDH) was crystallized from concentrated ammonium sulfate solution and cross-linked with glutaraldehyde to afford long-lived enzymatically active cross-linked crystals (LDH-CLC). The crystals were employed in an electrolytic cell for lactate production from pyruvate, in which the cathode consisted of a carbon electrode containing a coating of lipoamide dehydrogenase (LiDH) immobilized with methyl viologen under a Nafion membrane. This cell was more effective than a similar cell containing LDH in soluble form. An even greater improvement in performance was achieved by chemically binding a viologen derivative to the LiDH and using an electrode based on this modified enzyme in a cell containing LDH-CLC. The activity of the LDH-CLC is much less sensitive to pH than that of the soluble enzyme.