To answer the question, additional x-ray struc- 

 tures were obtained of the enzyme complexed 

 with "substrate analogues" (which are biochemi- 

 cally similar to the true substrate but are not rap- 

 idly digested by the enzyme). The studies showed 

 that the three-dimensional structure was flexible 

 in the presence of substrate, thus providing clear 

 evidence for the "induced fit" theory of enzyme 

 catalysis. The structure also provided valuable 

 information about the active site of the enzyme 

 and its mechanism of action. 



More recently. Dr. Michael G. Rossmann at 

 Purdue University, in his studies on lactate dehy- 

 drogenase, has identified a unique type of folding 

 that forms the binding site for a substrate com- 

 mon to all dehydrogenases. Other x-ray structural 

 investigations of this important class of enzymes 

 include the work of Dr. Leonard Banaszak on 

 malate dehydrogenase. 



Among the groups of enzymes being studied by 

 crystallographic methods are those involved in 

 regulating metabolic processes. In general, these 

 are very large structures, and progress in this area 

 has been slow. However, Dr. Thomas A. Steitz, 

 at Yale University, has obtained a high-resolu- 

 tion crystal structure for yeast hexokinase. This 

 work is providing insights into the subunit interac- 

 tions and regulator binding sites of a known regula- 

 tory enzyme. Other studies of this type, still in 

 progress, are being carried out by Dr. David S. 

 Eisenberg (University of California, Los Angeles) 

 on glutamine synthetase and Dr. William N. Lip- 

 scomb (Harvard University) on aspartyl transcar- 

 bamylase. 



The crystallographic methodology has been ap- 

 plied to a wide range of macromolecules with 

 some results of considerable medical importance. 

 For example, the structure of one of the macro- 

 molecules responsible for the immune response was 

 worked out at NIH by Dr. D. R. Davies and 

 co-workers. The resulting improved knowledge of 

 antibody structure has profoundly changed the 

 immunologist's view of how these macromole- 

 cules function in the whole immunogenic mechan- 

 ism. This may ultimately lead to the engineering 

 of macromolecules that respond to specific con- 

 taminants. 



Other macromolecule research with more imme- 

 diate medical payoff is the determination of the 

 structure of sickle hemoglobin by Dr. Warner E. 

 Love and his co-workers at Johns Hopkins Uni- 

 versity. A simple amino acid substitution in the 

 structure of the hemoglobin macromolecule, which 

 is the main component of blood, causes the blood 

 cells to distort from a flat to a sickle shape. 

 Crystallographic determinations of the subtle 

 shape changes in the hemoglobin are being used 

 to determine why the hemoglobin macromolecules 



pack in a different arrangement. Knowledge of 

 the conditions that bring about the onset of sickle 

 packing of the hemoglobin has led Dr. William A. 

 Eaton and co-workers at NIH to develop ap- 

 propriate screening techniques. 



Other major advances in the understanding of 

 protein structure and function have come from 

 the application of sophisticated biophysical tech- 

 niques. Dr. Peter Debrunner at the University of 

 Illinois has been studying the iron reaction center 

 of a cytochrome P450, utilizing primarily the tech- 

 niques of Mossbauer spectroscopy and electron 

 spin resonance. This protein is believed to be in- 

 volved in detoxification mechanisms in higher 

 organisms. Dr. Debrunner's studies provide evi- 

 dence of the state of the iron atom when com- 

 plexed with oxygen, the bound state of the mole- 

 cular oxygen, and the possible protein ligands that 

 interact with the iron. Eventually, it is hoped that 

 these studies, together with other approaches, will 

 lead to an understanding of how this protein func- 

 tions. 



Similarly, Dr. Lubert Stryer at Stanford Uni- 

 versity has applied Raman spectroscopy to the 

 study of the light-induced isomerization of the 

 visual pigment, rhodopsin. These studies should 

 aid in understanding how the visual pigment 

 changes following absorption of light. Studies on 

 a bacterial rhodopsin by Dr. Walter Stoeckenius 

 at the University of California at San Francisco 

 are making significant contributions toward under- 

 standing the way organisms respond to light. 



A major area of research is on subunit interac- 

 tions of enzymes that regulate metabolic process- 

 es. Dr. Howard K. Schachman, of the University 

 of California at Berkeley, has been studying the 

 nature of the subunit interactions that govern the 

 regulatory role of a bacterial enzyme, aspartate 

 transcarbamylase. Together with Dr. John C. 

 Gerhart, he has found that the enzyme contains 

 two discrete classes of subunits. One class catalyz- 

 es the enzymatic reaction, and the other class mo- 

 difies this reaction through its interaction with 

 small molecular weight activators and inhibitors. 

 Dr. Schachman and his co-workers have been 

 studying the various interactions between sub- 

 units, in hybrid species composed of native, chemi- 

 cally modified, and mutant chains, to determine 

 the molecular mechanism of regulation. Similar 

 studies are being carried out on the enzyme from 

 milk that catalyzes the synthesis of lactose. As 

 shown by Dr. Kurt Ebner in 1966-67, this enzyme 

 consists of a regulatory and a catalytic subunit. 

 The mechanism of regulation and the nature of the 

 subunit interactions are being studied by both Dr. 

 Ebner at the University of Kansas and Dr. Keith 

 Brew at the University of Miami. 



HEALTH, EDUCATION AND WELFARE 103 



