Past Times Regulars A 21st Century perspective on the contributions of

Michaelis to kinetics Downloaded from http://portlandpress.com/biochemist/article-pdf/34/2/47/3342/bio034020047.pdf by guest on 27 September 2021

Herbert Gutfreund Leonor Michaelis (1875–1949) made some of the most important contributions to the (University of Bristol, UK) application of physical chemistry to biological systems during the first half of the 20th Century. Like many young men interested in using basic physics and chemistry to study biomedical problems at that time, Michaelis was advised by no less a person than to qualify in medicine to be able to earn a living. He followed that advice, and the work I am concerned with here was carried out after he completed his medical studies. For about 5 years before the outbreak of World War I, Michaelis’s principal research interests centred on and the importance of hydrogen ions in biological systems. He carried out his basic research in clinical laboratories side by side with his medical work.

The first two of the following three topics of Michaelis investigations in enzyme kinetics are of principal interest for our present discussion. 1. The effects of hydrogen ion concentration on the rates of enzyme reactions 2. The effects of substrate concentration on the rates of enzyme reactions at constant pH. 3. Inhibitors of enzyme reactions that compete with substrate for binding to the enzyme (competitive inhibitors) and inhibitors that decrease the rate of the reaction of the enzyme–substrate complex to form product and free enzyme.

My concern here is to link Michaelis’s groundbreaking investigations to the development of these topics during the rest of the 20th Century. In both of them, his major contribution to the work of his contemporaries was the design of his experiments and the practical skill of their executions that provided conclusive evidence for quantitative laws. The Danish physical chemist Sorensen (1886– 1939) had just extended his studies of the effects of hydrogen ions on proteins to enzyme reactions1 when Michaelis began to investigate this topic in detail. Most of his results are summarized in his important Figure 1. Title page of Michaelis’s 1914 work monograph Die Wasserstoffionen Konzentrazion: Ihre Bedeutung fuer die Biologie und die Methoden reactions. The concept of buffers was also developed Ihrer Messung (Figure 1). An important conclusion for this purpose. was that the hydrogen ion concentration (defined on The data from Michaelis’s study2 of the pH- the logarithmic scale as pH) has to be maintained dependence of the rate of hydrolysis of peptone by constant during the study of other effects on enzyme trypsin shown in Figure 2a can be compared with

April 2012 © Biochemical Society 47 Regulars Past Times

those in Figure 2b which present the results of a a similar investigation using a well defined substrate (benzoyl-arginine ethyl ester) carried out about 40 years later3. I am filled with shame for not having referred to the earlier work of Michaelis that was carried out to establish the ionic nature of as proteins and the importance of pH constancy. The later investigation, with solutions of crystalline enzyme and the hydrolysis of one specific ester bond was designed to provide precise physicochemical data of one ionizing group of the enzyme. There is a remarkable agreement between the two studies Downloaded from http://portlandpress.com/biochemist/article-pdf/34/2/47/3342/bio034020047.pdf by guest on 27 September 2021 with respect to the involvement of an ionizing group

with pKa 6.2 to 6.3. Since the pKa refers to a group of trypsin at the active site with attached substrate, it is therefore dependent on the nature of the bound substrate, which differs in the two investigations. There could be severa