Pitfalls in the study of steady state kinetics of enzymes: spurious inhibition patterns due to stray light errors

When reaction velocity measurements of enzyme reactions are carried out with single beam, single monochromator spectrophotometers, stray light in the spectrophotometer can produce systematic errors in the apparent velocities when highly absorbing solutions (optical density >2.0) are used. These errors can give rise to spurious “inhibition” patterns of the steady state kinetics. Because of a suspected error of this kind, this laboratory has recently reinvestigated the kinetics of glucose 6-phosphate dehydrogenase from Escherichia coli and found that the reported noncompetitive inhibition of the enzyme by DPNH is explained more readily by an unnoticed effect of stray light on the apparent reaction velocity than by a true enzyme inhibition. Methods for estimating and correcting such errors in spectrophotometers are presented in detail.