Exploring and Engineering Lactate Dehydrogenase Examining the structure of an enzyme can lead to hypotheses about the relationship between different amino acids in the protein's structure and the protein's function. One way to test these hypotheses is to use recombinant DNA technology to generate mutant versions of the enzyme and then examine the structure and function of these altered forms. The technology used to do this is described in Chapters 8 and 9 One example of this kind of analysis is the work of A. R. Clarke and colleagues on the enzyme lactate dehydrogenase, published in $1989 .$ Lactate dehydrogenase (LDH) catalyzes the reduction of pyruvate with NADH to form lactate (see Section 14.3). A schematic of the enzyme's active site is shown below; the pyruvate is in the center:
The reaction mechanism is similar to that of many NADH reductions (see Fig. $13-24$ ); it is approximately the reverse of steps 2 and 3 of Figure $14-8 .$ The transition state involves a strongly polarized carbonyl group of the pyruvate molecule:
(a) A mutant form of LDH in which Arg $^{109}$ is replaced with Gln shows only $5 \%$ of the pyruvate binding and $0.07 \%$ of the activity of wild-type enzyme. Provide a plausible explanation for the effects of this mutation.
(b) A mutant form of LDH in which Arg $^{171}$ is replaced with Lys shows only $0.05 \%$ of the wild-type level of substrate binding. Why is this dramatic effect surprising?
(c) In the crystal structure of LDH, the guanidinium group of Arg $^{171}$ and the carboxyl group of pyruvate are aligned, as shown above, in a co-planar "forked" configuration. Based on this structure, explain the dramatic effect of substituting Arg 171 with Lys.
(d) A mutant form of LDH in which Ile $^{250}$ is replaced with Gln shows reduced binding of NADH. Provide a plausible explanation for this result.
Clarke and colleagues also set out to engineer a mutant version of LDH that would bind and reduce oxaloacetate rather than pyruvate. They made a single substitution, replacing $\mathrm{Gln}^{102}$ with Arg; the resulting enzyme would reduce oxaloacetate to malate and would no longer reduce pyruvate to lactate. They had therefore converted LDH to malate dehydrogenase.
(e) Sketch the active site of this mutant LDH with oxaloacetate bound.
(f) Why does this mutant enzyme now use oxaloacetate as a substrate instead of pyruvate?
(g) The authors were surprised that substituting a larger amino acid in the active site allowed a larger substrate to bind. Explain this result.